Methods and systems for extending dynamic range in assays for the detection of molecules or particles

ABSTRACT

Described herein are systems and methods for extending the dynamic range of assay methods and systems used for determining the concentration of analyte molecules or particles in a fluid sample. In some embodiments, a method comprises spatially segregating a plurality of analyte molecules in a fluid sample into a plurality of locations. At least a portion of the locations may be addressed to determine the percentage of said locations containing at least one analyte molecule. Based at least in part on the percentage, a measure of the concentration of analyte molecules in the fluid sample may be determined using an analog, intensity-based detection/analysis method/system and/or a digital detection/analysis method/system. In some cases, the assay may comprise the use of a plurality of capture objects.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/827,815, filed Aug. 17, 2015, and issued as U.S. Pat. No. 9,846,155,entitled “Methods and Systems for Extending Dynamic Range in Assays forthe Detection of Molecules or Particles,” which is a divisional of U.S.patent application Ser. No. 13/037,987, filed Mar. 1, 2011, and issuedas U.S. Pat. No. 9,110,025, entitled “Methods and Systems for ExtendingDynamic Range in Assays for the Detection of Molecules or Particles,” byRissin et al., which claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/441,894, filed Feb. 11, 2011, entitled “Methodsand Systems for Extending Dynamic Range in Assays for the Detection ofMolecules or Particles,” by Rissin et al. U.S. patent application Ser.No. 13/037,987 is a continuation-in-part of U.S. patent application Ser.No. 12/731,136, filed Mar. 24, 2010, and issued as U.S. Pat. No.8,415,171, entitled “Methods and Systems for Extending Dynamic Range inAssays for the Detection of Molecules or Particles,” by Rissin et al.,which claims the benefit of U.S. Provisional Patent Application Ser. No.61/309,165, filed Mar. 1, 2010, entitled “Methods and Systems forExtending Dynamic Range in Assays for the Detection of Molecules orParticles,” by Rissin et al. Each of the above-indicated applications isincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under contractR43CA133987 awarded by the National Cancer Institute. The government hascertain rights in the invention.

FIELD OF THE INVENTION

Described herein are systems and methods for extending the dynamic rangeof analytical assays and systems used for determining a concentration ofanalyte molecules or particles in a fluid sample.

BACKGROUND OF THE INVENTION

Methods and systems that are able to quickly and accurately detect and,in certain cases, quantify a target analyte molecule in a sample are thecornerstones of modern analytical measurements. Such systems and methodsare employed in many areas such as academic and industrial research,environmental assessment, food safety, medical diagnosis, and detectionof chemical, biological, and radiological warfare agents. Advantageousfeatures of such techniques may include specificity, speed, andsensitivity.

Many of the known methods and techniques are limited by the dynamicrange of the concentrations the methods and techniques can detectaccurately (e.g., limited dynamic range) and/or do not have thesensitivity to detect molecules or particles when they are present atvery low concentration.

Accordingly, improved systems and methods for extending the dynamicrange of analytical assays and systems used for determining a measure ofthe concentration of molecules or particles in a fluid sample areneeded.

SUMMARY OF THE INVENTION

Described herein are systems and methods for extending the dynamic rangeof analytical methods and systems used for determining the concentrationof analyte molecules or particles in a fluid sample. The subject matterof the present invention involves, in some cases, interrelated products,alternative solutions to a particular problem, and/or a plurality ofdifferent uses of one or more systems and/or articles.

In some embodiments, a system for determining a measure of theconcentration of analyte molecules or particles in a fluid sample,comprising an assay substrate comprising a plurality of locations eachcomprising a binding surface forming or contained within such locations,wherein at least one binding surface comprises at least one analytemolecule or particle immobilized on the binding surface, at least onedetector configured to address a plurality of the locations, able toproduce at least one signal indicative of the presence or absence of ananalyte molecule or particle at each location addressed and having anintensity varying with the number of analyte molecules or particles ateach location, and at least one signal processor configured to determinefrom the at least one signal the percentage of said locations containingat least one analyte molecule or particle, and further configured to,based upon the percentage, either determine a measure of theconcentration of analyte molecules or particles in the fluid samplebased at least in part on the number of locations containing at leastone analyte molecule or particle, or determine a measure of theconcentration of analyte molecules or particles in the fluid samplebased at least in part on an intensity level of the at least one signalindicative of the presence of a plurality of analyte molecules orparticles.

In some embodiments, a method for determining a measure of theconcentration of analyte molecules or particles in a fluid samplecomprises providing analyte molecules or particles immobilized withrespect to a binding surface having affinity for at least one type ofanalyte molecule or particle, the binding surface forming or containedwithin one of a plurality of locations on a substrate, addressing atleast some of the plurality of locations and determining a measureindicative of the percentage of said locations containing at least oneanalyte molecule or particle, and based upon the percentage, eitherdetermining a measure of the concentration of analyte molecules orparticles in the fluid sample based at least in part on the number oflocations containing at least one analyte molecule or particle ordetermining a measure of the concentration of analyte molecules orparticles in the fluid sample based at least in part on a measuredintensity of a signal that is indicative of the presence of a pluralityof analyte molecules or particles.

In some embodiments, a method for determining a measure of theconcentration of analyte molecules or particles in a fluid samplecomprises exposing a plurality of capture objects, each including abinding surface having affinity for at least one type of analytemolecule or particle, to a solution containing or suspected ofcontaining the at least one type of analyte molecules or particles,wherein at least some of the capture objects become associated with atleast one analyte molecule or particle, spatially segregating at least aportion of the capture objects subjected to the exposing step into aplurality of locations, addressing at least some of the plurality oflocations and determining a measure indicative of the percentage saidlocations containing a capture object associated with at least oneanalyte molecule or particle, wherein the locations addressed arelocations which contain at least one capture object, and based upon thepercentage, either determining a measure of the concentration of analytemolecules or particles in the fluid sample based at least in part on thenumber of locations containing a capture object associated with at leastone analyte molecule or particle, or determining a measure of theconcentration of analyte molecules or particles in the fluid samplebased at least in part on a measured intensity level of a signal of thatis indicative of the presence of a plurality of analyte molecules orparticles.

BRIEF DESCRIPTION OF THE DRAWINGS

Other aspects, embodiments, and features of the invention will becomeapparent from the following detailed description when considered inconjunction with the accompanying drawings. The accompanying figures areschematic and are not intended to be drawn to scale. For purposes ofclarity, not every component is labeled in every figure, nor is everycomponent of each embodiment of the invention shown where illustrationis not necessary to allow those of ordinary skill in the art tounderstand the invention. All patent applications and patents mentionedin the text are incorporated by reference in their entirety. In case ofconflict between the description contained in the present specificationand a document incorporated by reference, the present specification,including definitions, will control.

FIG. 1 shows a graphical representation of results of a Poissondistribution adjustment, as performed according to some embodiments;

FIG. 2 shows a graph of the average number of enzymes bound per beadversus the concentration of enzymes in a fluid sample, according to oneembodiment;

FIG. 3 shows a graph of the average number of enzymes bound per beadversus the concentration of enzymes in a fluid sample, according to oneembodiment, wherein the average number of enzymes bound per bead iscalculated using two different analytical methods;

FIG. 4A shows a plot of the fraction of active beads versus theeffective analyte concentration, given by the average molecule per bead(AMB), determined from digital counting using the Poisson distribution,accordingly to one embodiment;

FIG. 4B shows a plot of analog intensity (I_(bead)/I_(single)) as afunction of effective concentration, AMB, accordingly to one embodiment;

FIG. 4C shows plot of the imprecision in AMB (% CV) as a function of thenumber of active beads from (i) digital analysis and (ii) analoganalyses, according to one embodiment;

FIG. 5A shows a schematic diagram of an assay protocol of an embodimentof the present invention, wherein AMB=0.1 (left), AMB=0.6 (middle), andAMB=3;

FIGS. 5B-D show fluorescence images generated using an assay accordingto some embodiments of singulated beads in individual wells atapproximate AMBs of (D) 0.1, (E) 0.6, and (F) 3.0;

FIG. 6 shows a plot of the number of resorufin molecules produced as afunction of the number of enzymes on a bead, according to someembodiments;

FIG. 7 shows a graph of the fluorescence intensity versus time, whichmay be used to determine the rate of photobleaching, according to someembodiments;

FIG. 8 is a schematic diagram depicting one embodiment of a step of amethod of the invention employing a precursor labeling agent;

FIGS. 9A and 9B show a non-limiting example of a system employing anoptical detection system;

FIG. 10 is a schematic block diagram showing a system employing a fiberoptic assembly with an optical detection system;

FIG. 11 is a schematic flow diagram depicting an embodiment of a method(steps A-D) for the formation of a plurality of reaction vessels throughmating of a substrate and a sealing component and depicting examples ofthe size (E, F) of a sealing component relative to a substrate;

FIG. 12A depicts an experimental set-up for detection using light;

FIG. 12B shows a fiber optic array that has been sealed with a sealingcomponent;

FIG. 13A shows a plot of AMB as a function of enzyme concentration,according to one embodiment;

FIG. 13B shows a table including the % active beads and AMB values as afunction of enzyme concentration, according to one embodiment;

FIGS. 14A and 14B are plots showing a combined digital and analoganalysis of PSA samples, according to some embodiments;

FIG. 15A is a data table that shows conversion of % active beads toAMB_(digital) using Poisson statistics, according to one embodiment; and

FIG. 15B shows a plots of % active beads (diamonds) and AMB_(digital)(squares) as a function of enzyme concentration, according to someembodiments.

DETAILED DESCRIPTION

Described herein are systems and methods for extending the dynamic rangeof analytical assay methods and systems used for determining aconcentration of analyte molecules or particles (such as, for example,cells, cell organelles and other biological or non-biologicalparticulates) in a fluid sample. The subject matter of the presentinvention involves, in some cases, interrelated products, alternativesolutions to a particular problem, and/or a plurality of different usesof one or more systems and/or articles. It should be understood, thatwhile much of the discussion below is directed to analyte molecules,this is by way of example only, and other materials may be detectedand/or quantified, for example, analytes in particulate form. Someexemplary examples of analyte molecules and particles are describedherein.

The methods and system described herein may be useful to extend thedynamic range of analytical methods and systems used in certainembodiments by employing two or more techniques for determining ameasure of the concentration of analyte molecules in a fluid sample. Insome embodiments, the dynamic range may be extended by combining both ananalog, intensity-based detection/analysis method/system and a digitaldetection/analysis method/system, as described herein. In some cases,when the analyte molecules in the fluid sample are present at lowerconcentration ranges, single analyte molecules may be detected and thenumber of analyte molecules may be determined. A measure of theconcentration of analyte molecules in a fluid sample may be based atleast in part on this data (e.g., the number of analyte molecule) usinga digital analysis method/system. In some cases, the data may be furthermanipulated using a Poisson distribution adjustment. At higherconcentration ranges (e.g., at concentration levels whereisolating/detecting/determining single analyte molecules may become lesspractical) a measure of the concentration of analyte molecules in thefluid sample may be determined using and analog, intensity level-basedtechnique. In an analog analysis method/system, the measure of theconcentration may be based at least in part on a measured relativesignal intensity, wherein the total measured intensity may be correlatedwith the presence and quantity of analyte molecules. In certainembodiments, both analog and digital capability may be combined in asingle assay/system, such that, for example, a calibration standard maybe developed for an analyte molecule of interest across a wide dynamicrange. In one such example, a single calibration curve may be generatedusing both a digital and analog quantification technique, wherein thedigital and analog regimes of the calibration are linked by using acalibration factor, as described herein. The determination of an unknownconcentration of an analyte molecule in a test fluid sample may be basedat least in part by comparing test results (e.g., number/fraction oflocations containing an analyte molecule (digital) and/or measuredintensity level (analog)) with the calibration curve.

The term, “dynamic range” is given its ordinary meaning in the art andrefers to the range of the concentration of analyte molecules in a fluidsample that may be quantitated by a system or method without dilution orconcentration of the sample or change in the assay conditions producinga similar result (e.g., concentration of reagents employed, etc.), andwherein the measured concentration of the analyte molecules may besubstantially accurately determined. The concentration of analytemolecules in a fluid sample may be considered to be substantiallyaccurately determined if the measured concentration of the analytemolecules in the fluid sample is within about 10% of the actual (e.g.,true) concentration of the analyte molecules in the fluid sample. Incertain embodiments, the measured concentration of the analyte moleculesin the fluid sample is substantially accurately determined inembodiments where the measured concentration is within about 5%, withinabout 4%, within about 3%, within about 2%, within about 1%, withinabout 0.5%, within about 0.4%, within about 0.3%, within about 0.2%, orwithin about 0.1% of the actual concentration of the analyte moleculesin the fluid sample. In some cases, the measure of the concentrationdetermined differs from the true (e.g., actual) concentration by nogreater than about 20%, no greater than about 15%, no greater than about10%, no greater than about 5%, no greater than about 4%, no greater thanabout 3%, no greater than about 2%, no greater than about 1%, or nogreater than about 0.5%. The accuracy of the assay method may bedetermined, in some embodiments, by determining the concentration ofanalyte molecules in a fluid sample of a known concentration using theselected assay method and comparing the measured concentration with theactual concentration.

In some embodiments, the inventive systems or methods may be capable ofmeasuring concentrations of analyte molecules in a fluid sample over adynamic range of more than about 1000 (3 log), about 10,000 (4 log),about 100,000 (5 log), about 350,000 (5.5 log), 1,000,000 (6 log), about3,500,000 (6.5 log), about 10,000,000 (7 log), about 35,000,000 (7.5log), about 100,000,000 (8 log), or more.

In some embodiments, the concentration (e.g., unknown concentration) ofanalyte molecules in the fluid sample that may be substantiallyaccurately determined is less than about 5000 fM (femtomolar), less thanabout 3000 fM, less than about 2000 fM, less than about 1000 fM, lessthan about 500 fM, less than about 300 fM, less than about 200 fM, lessthan about 100 fM, less than about 50 fM, less than about 25 fM, lessthan about 10 fM, less than about 5 fM, less than about 2 fM, less thanabout 1 fM, less than about 500 aM (attomolar), less than about 100 aM,less than about 10 aM, less than about 5 aM, less than about 1 aM, lessthan about 0.1 aM, less than about 500 zM (zeptomolar), less than about100 zM, less than about 10 zM, less than about 5 zM, less than about 1zM, less than about 0.1 zM, or less. In some cases, the limit ofdetection (e.g., the lowest concentration of an analyte molecule whichmay be determined in solution) is about 100 fM, about 50 fM, about 25fM, about 10 fM, about 5 fM, about 2 fM, about 1 fM, about 500 aM(attomolar), about 100 aM, about 50 aM, about 10 aM, about 5 aM, about 1aM, about 0.1 aM, about 500 zM (zeptomolar), about 100 zM, about 50 zM,about 10 zM, about 5 zM, about 1 zM, about 0.1 zM, or less. In someembodiments, the concentration of analyte molecules or particles in thefluid sample that may be substantially accurately determined is betweenabout 5000 fM and about 0.1 fM, between about 3000 fM and about 0.1 fM,between about 1000 fM and about 0.1 fM, between about 1000 fM and about0.1 zM, between about 100 fM and about 1 zM, between about 100 aM andabout 0.1 zM, or less. The upper limit of detection (e.g., the upperconcentration of an analyte molecule which may be determined insolution) is at least about 100 fM, at least about 1000 fM, at leastabout 10 pM (picomolar), at least about 100 pM, at least about 100 pM,at least about 10 nM (nanomolar), at least about 100 nM, at least about1000 nM, at least about 10 uM, at least about 100 uM, at least about1000 uM, at least about 10 mM, at least about 100 mM, at least about1000 mM, or greater. In some embodiments, the concentration of analytemolecules or particles in the fluid sample determined is less than about50×10⁻¹⁵ M, or less than about 40×10⁻¹⁵ M, or less than about 30×10⁻¹⁵M, or less than about 20×10⁻¹⁵ M, or less than about 10×10⁻¹⁵ M, or lessthan about, or less than about 1×10⁻¹⁵ M.

Exemplary Combined Digital/Analog Analysis Methods/Systems

The following section describes exemplary methods and systems forextending the dynamic range of analytical methods/systems used todetermine a measure of concentration of analyte molecules or particlesin a fluid sample. In some embodiments, the analytical method employedis capable of individually isolating and detecting single analytemolecules at low concentrations. In some cases, the analytical methodinvolves spatially segregating a plurality of analyte molecules into aplurality of locations in or on a surface of a substrate (e.g., plate,chip, optical fiber face, etc.). At low concentration ranges, theanalyte molecules may be spatially segregated such that a statisticallysignificant fraction of such locations contain no analyte molecules withat least some of the locations containing at least one analyte molecule.Methods and systems which may be used in conjunction with themethods/systems of the present invention for extending the dynamic rangeare described herein.

As an exemplary method, and as described in more detail herein, aplurality of analyte molecules in a fluid sample may be made to becomeimmobilized with respect to a plurality of capture objects (e.g., beads)that each include a binding surface having affinity for at least onetype of analyte molecule (see, for example, methods and capture objectsdescribed in commonly owned U.S. patent application Ser. No. 12/731,130,entitled “Ultra-Sensitive Detection of Molecules or Particles usingBeads or Other Capture Objects” by Duffy et al., filed Mar. 24, 2010;and International. Patent Application No. PCT/US11/026,645, entitled“Ultra-Sensitive Detection of Molecules or Particles using Beads orOther Capture Objects” by Duffy et al., filed Mar. 1, 2011, each hereinincorporated by reference). At least some of the beads (e.g., at leastsome associated with at least one analyte molecule) may be spatiallyseparated/segregated into a plurality of locations (e.g., reactionvessels), and at least some of the reaction vessels may beaddressed/interrogated to detect the presence of a bead and analytemolecule. In some cases, the plurality of reaction vessels addressed isa portion or essentially all of the total quantity of reaction vesselswhich may contain at least one capture object (e.g., either associatedwith at least one analyte molecule or not associated with any analytemolecules). It should be understood, that while much of the discussionherein focuses on methods comprising immobilizing analyte molecules withrespect to beads (or other capture objects) prior to spatiallysegregating the plurality of analyte molecules into a plurality ofreaction vessels, this is by no means limiting, and othermethods/systems may be used for spatially segregating the analytemolecules, (e.g., where the analyte molecules are segregated into aplurality of locations without being immobilized on capture objects).Those of ordinary skill in the art will be able to apply the methods,systems, and analysis described herein to methods which do not employcapture objects (e.g., beads). For example, see U.S. Patent ApplicationNo. 20070259448, entitled “Methods and arrays for target analytedetection and determination of target analyte concentration insolution,” by Walt et al., filed Feb. 16, 2007; U.S. Patent ApplicationNo. 20070259385, entitled “Methods and arrays for detecting cells andcellular components in small defined volumes,” by Walt et al., filedFeb. 16, 2007; U.S. Patent Application No. 20070259381, entitled“Methods and arrays for target analyte detection and determination ofreaction components that affect a reaction” by Walt et al., filed Feb.16, 2007; International Patent Application No. PCT/US07/019184, entitled“Methods for Determining the Concentration of an Analyte in Solution” byWalt et al., filed Aug. 20, 2007; and International Patent ApplicationNo. PCT/US09/005428, entitled “Ultra-Sensitive Detection of Molecules orEnzymes” by Duffy et al., filed Sep. 9, 2009, herein incorporated byreference.

Following spatially segregating the beads into the reaction vessels, atleast a portion of the reaction vessels may be addressed/interrogated todetermine the number and/or percentage of the locations addressed whichcontain a bead associated with at least one analyte molecule. In somecases, the locations addressed are at least a portion of the locationswhich contain at least one bead (e.g., either associated with at leastone analyte molecule or not associated with any analyte molecules). Thepercentage of locations which contain a bead associated with at leastone analyte molecule (the percentage of “active” beads) is the number ofbeads associated with at least one analyte molecule divided by the totalnumber of beads addressed, multiplied by 100%. Alternatively, ifdesired, the percentage of activity may be based on the number oflocations addressed whether or not they contain a bead (i.e. active beadcontaining locations as a percentage of locations addressed). As will beunderstood by those of ordinary skill in the art, in embodiments wherebeads (or other capture objects) are not employed, the percentage“active beads” in the following discussion may be substituted for thepercentage of locations containing at least one analyte molecule (e.g.,the percentage “active locations”).

In some embodiments, by determining the number/percentage of activebeads the bulk analyte concentration in the fluid sample can bedetermined. Particularly at low concentration levels (e.g., in thedigital concentration range), a measure of the concentration of analytemolecules in a fluid sample may be determined at least in part bycounting beads as either “on” (e.g., a reaction vessel containing a beadassociated with at least one analyte molecule) or “off” (e.g., areaction vessel containing a bead not associated with any analytemolecule). At low ratios of analyte molecules to beads (e.g., less thanabout 1:5, less than about 1:10, less than about 1:20, or less), nearlyall of the beads are associated with either zero or one analytemolecule. In this range, the percentage of active beads (e.g., “on”reaction vessels) may increase substantially linearly with increasinganalyte concentration, and a digital analysis method may beadvantageously used to analyze the data.

As the analyte concentration increases, however, a significantpopulation of the beads generally associate with more than one analytemolecule. That is, at least some of the beads associate with two, three,etc. analyte molecules. Therefore, as the analyte concentrationincreases, at some point the percentage of active beads in a populationgenerally will not be as linearly related to the bulk analyteconcentration since a greater fraction of the beads may associate withmore than one analyte molecule. In these concentration ranges, the datamay still be advantageously analyzed using a digital analysis method(e.g., counting “on” and “off” beads), however it may be possible toimprove the accuracy of the assay by applying a Poisson distributionadjustment to account for the binding probability of a population ofanalyte molecules to a population of beads. For example, according toPoisson distribution adjustment, in an assay that reports about 1.0%active beads (e.g., the ratio of beads associated with at least oneanalyte molecule to the total number of beads is about 1:100), about 99%of the beads are free of analyte molecules, about 0.995% of beadsassociate with one analyte molecule, and about 0.005% of beads associatewith two analyte molecules. As a comparison, in an assay that reportsabout 20.0% active beads (e.g., the ratio of beads associated with atleast one analyte molecule to the total number of beads is about 1:5),about 80% of the beads are free of analyte molecules, about 17.85% ofbeads associate with one analyte molecule, about 2.0% of beadsassociated with two analyte molecules and about 0.15% of beadsassociated with three analyte molecules. The non-linear effect (e.g., asseen in the second comparative example) can be accounted for across theentire concentration range in which there remains a statisticallysignificant fraction (e.g., as described herein—see Equation 1 below andassociated discussion) of beads not associated with any analytemolecules or particles in the sample (e.g., the range in which a digitalanalysis methods/system may be able to accurately determine a measure ofthe concentration, e.g., in some cases up to about 20% active beads, upto about 30% active beads, up to about 35% active beads, up to about 40%active beads, up to about 45% active beads, up to about 50% activebeads, up to about 60% active bead, up to about 70% active beads, ormore) using a Poisson distribution adjustment. A Poisson distributiondescribes the likelihood of a number of events occurring if the averagenumber of events is known. If the expected number of occurrences is a,then the probability (P_(μ)(ν)) that there are exactly ν occurrences (νbeing a non-negative integer, ν=0, 1, 2, . . . ) may be determined byEquation 1:

$\begin{matrix}{{P_{\mu}(v)} = {e^{- \mu}\left( \frac{\mu^{v}}{v!} \right)}} & \left( {{Eq}.\mspace{14mu} 1} \right)\end{matrix}$In some embodiments of the present invention, μ is equal to the ratio ofnumber of analyte molecules detected to the total number of beadsdetected (e.g., either associated with or not associated with anyanalyte molecules), and ν is the number of beads containing a certainnumber of analyte molecules (e.g., the number of beads associated witheither 0, 1, 2, 3, etc. analyte molecules). By determining μ from anexperiment, therefore, the number and, through further calculations, theconcentration of analyte molecules can be determined. In thedigital/binary mode of measurements where beads associated with 1, 2, 3,4, etc. analyte molecules are indistinguishable (e.g., where ν=1, 2, 3,4 are indistinguishable) and the analyte molecule containing beads (orlocations) are simply characterized as “on.” Occurrences of ν=0 can bydetermined definitively as the number of “off” beads (or locations).(P_(μ)(0)) may be calculated according to Equation 2:

$\begin{matrix}{{P_{\mu}(0)} = {{e^{- \mu}\left( \frac{\mu^{0}}{0!} \right)} = e^{- \mu}}} & \left( {{Eq}.\mspace{14mu} 2} \right)\end{matrix}$and the number of expected occurrences, μ, may be determined based on arearrangement of Equation 2, as given in Equation 3:μ=−ln [P _(μ)(0)]  (Eq. 3).

The number of occurrences of beads associated with no analyte molecules,P_(μ)(0), is equal to 1 minus the total number of beads with all otheroccurrences (e.g., beads associated at least one analyte molecule) thenμ is given by Equation 4:

$\begin{matrix}\begin{matrix}{\mu = \frac{{Number}\mspace{14mu}{of}\mspace{14mu}{analyte}\mspace{14mu}{molecules}}{{Total}\mspace{14mu}{number}\mspace{14mu}{of}\mspace{14mu}{beads}}} \\{= {- {{\ln\left( {1 - {{fraction}\mspace{14mu}{{of}\mspace{14mu}}^{``}{on}^{''}\mspace{14mu}{beads}}} \right)}.}}}\end{matrix} & \left( {{Eq}.\mspace{14mu} 4} \right)\end{matrix}$In some cases, μ is also referred to herein as “AMB_(digital).”Rearranging Equation 4, the total number of analyte molecules in thefluid sample contained in the counted locations can be determined usingEquation 5:Number of analyte molecules=−ln(1−fraction of “on” beads)×(Total numberof beads)  (Eq. 5).Therefore, the total number of molecules can be determined from thefraction of “on” beads for a given number of beads, and a measure of theconcentration of analyte molecules in the fluid sample may be based atleast in part on this number (as well as, e.g., any dilutions of thesample during the assay, the number and volume of the wells containingcapture objects interrogated, etc). The number of beads with 1, 2, 3, 4,etc. associated analyte molecules can also be determined by calculatingP_(μ)(1), P_(μ)(2), P_(μ)(3) etc. from the μ determined and Equation 1.

Table 1 demonstrates the potential utility of Poisson distributionadjustment. Column A shows the number of analyte molecules in the samplecalculated from the molarity and volume tested. Column B shows theunadjusted number of molecules captured on beads, where any beadassociated with any number (e.g., one, two, three, etc.) of analytemolecules is counted as being associated with a single analyte molecule.Column D is the Poisson adjusted data, wherein beads associated with twoanalyte molecules are counted as having two molecules bound, and beadsassociated with three molecules are counted as having three moleculesbound, etc. The comparison of the unadjusted and adjusted data can beseen by comparing Columns C and E. These columns give the calculatedcapture efficiencies of the assay at each concentration, wherein thecapture efficiency is the determined number of analyte moleculescaptured (unadjusted or Poisson adjusted) divided by the number ofanalyte molecules provided in the fluid sample, multiplied by 100%.Column C shows a calculation the capture efficiency using unadjusteddata, and a reduction in capture efficiency is observed as theconcentration of analyte molecules increases. Column E shows acalculation the capture efficiency using Poisson adjusted data. FIG. 1is a graphical representation of the results of an exemplary Poissondistribution adjustment. The unadjusted data deviates from linearitywith increasing concentration, while the data which has been subject toa Poisson distribution adjustment is substantially linear through asubstantially portion of the plotted concentration range. Using theresults shown in column D, the average number of analyte molecules perbead can be calculated (e.g., the Poisson adjusted number of moleculescaptured divided by the total number of beads addressed). In certainembodiments, the resulting average number of analyte molecules per beadmay be used to prepare a calibration curve, as described herein.

TABLE 1 Poisson distribution adjustment Column D Column A Column BColumn C Poisson Column E # of Unadjusted Digital Read adjusted PoissonMolecules # of Unadjusted # of Adjusted [SβG] in the Molecules CaptureMolecules Capture (aM) System Captured Efficiency Captured Efficiency0.35 21 28 132%  28 132%  0.7 42 33 79% 33 79% 3.5 211 159 75% 159 75% 7421 279 66% 279 66% 35 2107 1778 84% 1782 85% 70 4214 3267 78% 3280 78%350 21070 13514 64% 13748 65% 700 42140 30339 72% 31552 75% 3500 210700122585 58% 146380 69% 7000 421400 178112 42% 235716 56%

Above a certain active bead percentage (i.e., where there is no longer astatistically significant fraction of beads present in the populationthat are not associated with any of analyte molecules or particles, or,potentially advantageously for situations where there may be astatistically significant fraction of beads present in the populationthat are not associated with any of analyte molecules or particles butthat result in active bead percentages above a certain level—e.g.,greater than or substantially greater than about 40%, or about 50%, orabout 60%, or about 70% (or active location percentage, in embodimentswhere beads are not employed)) improvements in accuracy and/orreliability in the determination of analyte molecule concentration maypotentially be realized by employing an intensity measurement basedanalog determination and analysis rather than or supplementary to adigital/binary counting/Poisson distribution adjustment as previouslydescribed. At higher active bead percentages, the probability of anactive bead (e.g., positive reaction vessel) being surrounded by otheractive beads (e.g., positive reaction vessels) is higher and may incertain assay set ups create certain practical challenges to exclusivelyusing the digital/binary determination method. For example, in certainembodiments, leakage of a detectable component into a reaction vesselfrom an adjacent reaction vessel may occur to some extent. Use of ananalog, intensity level based technique in such situations canpotentially yield more favorable performance.

FIG. 2 illustrates a feature of the Poisson adjusted digital readouttechnique which may be observed, in some cases, as the analyteconcentration increases such that the number of active beads increasesto higher levels. At some point, in certain embodiments, theconcentration of analyte molecules may reach a level where the digitalreadout technique, with or without Poisson distribution adjustment, isno longer producing as linear a relationship with respect toconcentration as may be desirable, and the analytical technique employedby the system/method of the invention may be altered such that an analoganalysis method/system is employed. In analog analysis, the associationof multiple analyte molecules at high concentrations with single beadsmay able to be more effectively and/or reliably quantified. Theintensity of at least one signal from the plurality of reaction vesselswhich contains at least one analyte molecule may be determined. In somecases, the intensity determined may be the total overall intensitydetermined from all the reaction vessels interrogated containing atleast one analyte molecule (e.g., the intensity of the reaction vesselsis determined as a whole). In other cases, the intensity of eachreaction vessel producing a signal may be determined and averaged,giving rise to an average bead signal (ABS).

To extend the dynamic range of assay methods/systems of the invention tocombine both analog and digital analysis methods/systems, a “link” maybe established relating the results/parameters of the two analysismethods/systems. This may be done, in certain cases, with the aid of acalibration curve. In some embodiments, a measure of the unknownconcentration of analyte molecules in a fluid sample (e.g., test sample)may be determined at least in part by comparison of a measured parameterto a calibration curve, wherein the calibration curve includes datapoints covering both digital and analog concentration ranges, and hence,has an extended dynamic range as compared to a single mode (i.e., onlydigital or only analog) analysis method/system. The calibration curvemay be produced by conducting the assay with a plurality of standardizedsamples of known concentration under conditions substantially similar tothose used to analyze a test sample of unknown concentration. In oneexample, the calibration curve may transition from data determined usingan analog measurement to a digital analysis system/method as thedetected percentage of active beads is reduced to at or below athreshold value (e.g., about 40% active beads, or about 50% activebeads, or about 60% active beads, or about 70% active beads, etc.).

To prepare a combined digital-analog calibration curve, in certainembodiments a linkage is made between the results obtained in the lowconcentration (digital) and high concentration (analog) analyticalregimes. In certain embodiments, calibration curve relates analytemolecule concentration to a parameter defined as the average number ofanalyte molecules per bead, or AMB versus the concentration of moleculesin solution. It should be understood, that while the followingdiscussion features exemplary embodiments in which analyte moleculeshappen to be an enzyme, this is no means limiting, and in otherembodiments, other types of analyte molecules or particles may beemployed. For example, the analyte molecule may be a biomolecule, andthe assay may involve the use of a binding ligand which comprises anenzymatic component. The AMB for a sample with a concentration fallingin a range where digital analysis is preferred may be determined using aPoisson distribution adjustment, as described above. The AMB for asample with a concentration falling in a range where analog analysis ispreferred may be determined by converting an analog intensity signal(e.g., average bead signal) to an AMB using a conversion factor, asdiscussed below.

In a first exemplary embodiment, to prepare a calibration curve anddetermine an appropriate conversion factor, the assay is carried out ona calibration sample, wherein the percentage of active beads (orpercentage of active locations, in embodiments where beads are notemployed) is between about 30% and about 50%, or between about 35% andabout 45%, or in some cases about 40%, or in some cases greater than50%. The AMB for this sample can be calculated using a Poissondistribution adjustment, as described above. For this sample, theaverage bead signal (ABS) is also determined. A conversion factor (CF)relating the ABS and AMB may be defined, for example as according toEquation 6:

$\begin{matrix}{{C\; F} = {\frac{{AMB}_{{calibration}\mspace{14mu}{sample}}}{{ABS}_{{calibration}\mspace{14mu}{sample}}}.}} & \left( {{Eq}.\mspace{14mu} 6} \right)\end{matrix}$Therefore, the regime where it is preferred to use an analogdetermination, the AMB for a sample (e.g., unknown sample, or acalibration sample with a concentration placing it in the analog region)may be calculated according to Equation 7:AMB_(sample X)=CF×ABS_(sample X)  (Eq. 7).For example, in one exemplary embodiment, a calibration curve andconversion factor may be determined as follows. In Table 2 shown below,the highest concentration of analyte molecules (e.g., enzymes in thisexemplary embodiment) determined using a digital/binary read-outprotocol (about 7 fM) gave rise to about 42.36% active wells. Thisdigital signal was adjusted using Poisson distribution adjustment todetermine the total number of bound molecules, and the AMB wasdetermined to be 0.551. The data collected at this concentration levelwas also analyzed to determine the average bead signal (the ABS wasequal to about 1041 fluorescent units). The analog-to-digital conversionfactor was, therefore, 0.000529 AMB/fluorescent units, as calculatedusing Equation 6. For other samples with relatively high concentrationranges, the AMB value can be determined by applying this conversionfactor (e.g., as described in Equation 7). Table 2 illustrates theconversion of average bead signal (e.g., average beaded well intensityin Table 2) to AMB using an analog to digital conversion factor, as wellas the digitally determined AMB values at some lower concentrations. Thecombined digital/analog calibration data can be plotted on a singlecalibration curve of AMB values versus concentration. FIG. 3 shows agraphical representation of converted analog data plotted together withdigital data. The analog values are converted to AMB (data points 14)and plotted along with AMB values in the Poisson adjusted digitalreadout range (data points 12).

TABLE 2 Conversion of the analog readout to AMB Analog Converted DigitalPoisson Avg Beaded to Poisson ‘on’ Adjusted Well Intensity Adjusted AMBusing Combined fM percentage Digital AMB (Analog Measure) conversionfactor AMB ANALOG 700 — — 17243 9.130 9.130 350 — — 13996 7.411 7.411 70— — 5178 2.742 2.742 35 — — 3279 1.736 1.736 DIGITAL 7 42.36% 0.551 1041— 0.551 3.5 26.21% 0.304 — — 0.304 0.7 5.24% 0.054 — — 0.054 0.35 2.93%0.030 — — 0.030

The dynamic range demonstrated by the described approach based on theexperiments tabulated in Tables 1 and 2 was greater than 6 logs.Generally, the dynamic range of an analysis system/method is bounded bythe lower limit of detection for digital readout (e.g., in the specificexample described, about 227 zM) and the highest concentration testedand accurately quantifiable by analog readout (e.g., in this example,about 700 fM), i.e., 6.5 log. This dynamic range may be compared to thedynamic range of about 3 logs which can be achieved for the same testsamples on a plate reader that only has the ability to measure an analogsignal from an ensemble of molecules.

In a second exemplary embodiment, at low ratios of analyte molecules tobeads, where there are a significant number of beads that are associatedwith no analyte molecules (“off” beads), the number of active, analytemolecules-associated (or “on”) beads relative to the total number ofbeads detected may be are used to determine an AMB (i.e., AMB_(digital))via Poisson statistics as described above. At higher ratios, however, amodified approach is taken from that described above in the firstexemplary embodiment for preparing a calibration curve. In thisembodiment, at higher ratios of analyte molecules to beads, when mostbeads have one or more analyte molecules bound, and the countingapproach becomes less accurate, an AMB (i.e., AMB_(analog)) isdetermined from the average fluorescence intensity of wells containing abead in the array (Ī_(bead)). To convert Ī_(bead) to AMB in the analogregime, images with <10% active beads may be used to determine theaverage analog intensity of a single enzyme molecule (Ī_(single)). Theratio of Ī_(bead) to Ī_(single) over all beads provides an analog AMB,and a calibration curve can prepared as follows. The dynamic range of anassay may be extended beyond the digital regime by measuring the averagefluorescence intensity of wells that contain beads to determine thenumber of molecules (e.g., enzymes) associated with each bead detected.In this embodiment, the AMB can be determined from the averagefluorescence intensity value of the active beads (Ī_(bead)) and theaverage fluorescence intensity generated by a single bead (e.g., asingle enzyme; Ī_(single)). The AMB of an array in the analog range(AMB_(analog)) is defined by Equation 8:

$\begin{matrix}{{AMB}_{analog} = \frac{f_{on} \times {\overset{\_}{I}}_{bead}}{{\overset{\_}{I}}_{single}}} & \left( {{Eq}.\mspace{14mu} 8} \right)\end{matrix}$To determine Ī_(single), the AMB_(digital) (e.g., see Equation 4) andAMB_(analog) (Equation 8) can be equated in terms at fractions of activebeads where the beads predominantly associate with either one or zeromolecules dominate, for example, as shown in Equation 9. In some cases,these values are equated when there is negligible contribution fromsubstrate depletion (e.g., as described herein). In some cases, an arrayis analyzed and if the fractions of “on” beads <0.1 the condition istaken as meeting these criteria:

$\begin{matrix}{{{\overset{\_}{I}}_{single} = \frac{f_{on} \times {\overset{\_}{I}}_{bead}}{- {\ln\left\lbrack {1 - f_{on}} \right\rbrack}}},{{{in}\mspace{14mu}{arrays}\mspace{14mu}{where}\mspace{14mu} f_{on}} < {0.1.}}} & \left( {{Eq}.\mspace{14mu} 9} \right)\end{matrix}$AMB can then plotted for both the digital (AMB_(digital) (Equation 4))and analog (AMB_(analog) (Equation 8)) ranges, and the two curves may becombined into one calibration curve.

When combining the digital and analog data in this second embodiment, anexperiment may be employed to determine Ī_(single). The experiment mayemploy a sample wherein the fraction of active beads is less than about5%, about 10%, about 15%, about 20%, about 25%, or more. In some cases,the fraction of active beads is about 10%. This may accomplished, asdescribed above, by using calibration data points which cover thisrange, or specific control samples known to have a digital signal inthis range. With two or three concentrations in a calibration curve withf_(on)<0.1, the intensity of individual beads (e.g., the kineticactivities of individual enzyme molecules) can be averaged to determineĪ_(single). In the case of enzymes, the averaging of the intrinsicvariation associated with single enzyme molecule velocities (e.g., theenzyme turnover rate) may be such that little to no significantvariation to the Ī_(single) measurement is observed. The uncertainty inthe mean single enzyme intensity (Ī_(single)) as a function of Nmeasurements can be given by σ₁ _(single) /√{square root over (N)},where σ₁ _(single) is the width parameter of the normally distributedsingle enzyme molecule intensities. For example, with a width parameterof 30% of the average single enzyme velocities, the uncertainty added tothe mean value Ī_(single) was 1% when averaging over 1000 singlemolecule measurements. When the fraction of active beads increases(e.g., 10% and greater), theoretically both digital counting and analogintensities could be used to determine AMB. Below a certain percentageof active beads (e.g., less than about 20%, less than about 15%, lessthan about 10%, less than about 5%, or less), the contribution of beadsassociated with multiple enzymes may be too small such that Ī_(bead)does not vary above the measurement noise of % active beads and theanalog approach may not provide accurate results.

As f_(on) approaches 100%, as described above, counting “on” and “off”beads may not provide an accurate measurement of the AMB. Atintermediate percentages of “on” beads, various factors may beconsidered to determine the threshold of the fraction of active beadsbelow which AMB_(digital) (Equation 4) is used and above whichAMB_(analog)(Equation 8) is used. The choice of this threshold may beillustrated by plotting the imprecision in AMB arising from thevariation in digital and analog signals.

For example, FIG. 4A shows a plot of fraction of active beads againstthe effective concentration, given by AMB, determined from digitalcounting using the Poisson distribution (Equation 4). As concentrationincreases, the slope of % active gets shallower and signal imprecisionleads to greater imprecision in concentration determined. FIG. 4B showsa plot of analog intensity (I_(bead)/I_(single)) as a function ofeffective concentration, AMB (Equation 8). At low concentrations,variation in intensity measurements can make it difficult to detectsmall increases in multiple enzymes, and CVs of extrapolated AMB arehigh. FIG. 4C shows a plot of the imprecision in AMB (% CV) as afunction of f_(on) from (i) digital and (ii) analog analyses assuming afixed signal CV of 7.1% for both methods. In some cases, thedigital-to-analog threshold (e.g., the threshold where there is atransition of determining the concentration between using a digitalanalysis (e.g., Equation 4) or an analog analysis (e.g., Equation 8) isabout 40%, about 50%, about 60%, about 70%, about 80%, or between about50% and about 80%, or between about 60% and about 80%, or between about65% and about 75%. In a particular embodiment, the threshold is about70%, or between about 75% and about 85%. See Examples 8 and 9 for sampleexperiments.

FIG. 5 depicts an assay where determinations are made at varying AMBs.FIG. 5A (left) shows an AMB=0.1, wherein each active beads arestatistically associated predominantly with a single analyte moleculeand digital analysis may be conducted. FIG. 5A (middle) shows anAMB=0.6, wherein significant number of active beads are associated withmore than one analyte molecule, and an analog or digital analysis may beconducted. If a digital analysis is conducted, multiple analytemolecules per bead may be accounted for using a Poisson distributionanalysis. FIG. 5A (right) shows an AMB=3, wherein substantially all ofthe beads are associated with more than one analyte molecule. In thiscase, the average number of analyte molecules per bead may be quantifiedby measurement of the average fluorescence intensity of the active beadsand from knowledge of the average fluorescence intensity generated by asingle analyte molecules (e.g., enzyme), as described herein. FIG. 5B-Dshow fluorescence images generated using an assay as described herein ofsingulated beads in individual wells at approximate AMBs of (D) 0.1, (E)0.6, and (F) 3.0.

Once a calibration curve has been developed which relates the AMB to ameasure of the concentration of analyte molecules in a fluid sample, ameasure of concentration of analyte molecules in a test sample (e.g., anunknown sample) may be determined using the calibration curve. An assaymay be carried out in a similar manner as was conducted for thecalibration samples (e.g., including immobilizing the analyte moleculeswith respect to a plurality of beads, and spatially segregating at leasta portion of the plurality of beads into a plurality of reactionvessels). Following spatially segregating a plurality of beads into aplurality of reaction vessels, at least a portion of the plurality ofreaction vessels may be interrogated, in certain embodiments a pluralityof times. For example, at least about 1, at least about 2, at leastabout 3, at least about 4, at least about 5, at least about 6, at leastabout 7, at least about 8, at least about 9, at least about 10, or more,interrogations may be conducted, the interrogations separated by aperiod of time of, for example, about 1 second, about 2 seconds, about 5seconds, about 10 seconds, about 20 seconds, about 30 seconds, about 40seconds, about 50 seconds, about 1 minute, or more. Each interrogationmay produce one set of data. The data may be analyzed to determine thepercentage of beads associated with at least one analyte molecule (e.g.,the percentage active beads) (or the percentage of active locations, forexample, in embodiments where beads are not employed).

In some embodiments, if the percentage of active beads (or locations) isless than about 80%, less than about 70%, less than about 60%, less thanabout 50%, less than about 45%, less than about 40%, less than about35%, less than about 30%, less than about 25%, or less than about 20%,the measure of the concentration of analyte molecules or particles inthe fluid sample may be based at least in part on the number/percentageof locations determined to contain at least one analyte molecule orparticle. That is, at least one set of data may be analyzed using adigital analysis method (which may further include adjustment via aPoisson distribution adjustment) as described herein. For example, theAMB may be determined as described herein (e.g., using a Poissondistribution adjustment) and the concentration may be determined bycomparison of the AMB to the calibration curve. In some cases, the setof data used may be a data set collected later in time (e.g., to ensuresufficient time for an enzymatic substrate to be converted to adetectable entity). The measure of the concentration of analytemolecules in a fluid sample may be based at least in part on comparisonof a measured parameter to a calibration curve (e.g., formed at least inpart by determination of at least one calibration factor).

In other embodiments, for example if the percentage of active beads isrelatively high, e.g., greater than about 30%, greater than about 35%,greater than about 40%, greater than about 45%, greater than about 50%,greater than about 60%, greater than about 70%, greater than about 80%,or more, the measure of the concentration of analyte molecules orparticles in the fluid sample may be based at least in part onmeasurement of an intensity level of the at least one signal indicativeof the presence of a plurality of analyte molecules or particles. Thatis, the data may be analyzed using one of the analog analysis methods,as described herein. For example, the AMB may be determined for thesample using Equation 7 or Equation 8. The AMB may be compared with thecalibration curve to determine a measure of the concentration of analytemolecules in the fluid sample. In some cases the set of data used may bea data set collected earlier in time (e.g., such as to limitdifficulties associated with substrate depletion, photobleaching, etc.,as described herein). The measure of the concentration of analytemolecules in a fluid sample may be based at least in part on comparisonof a measured parameter to a calibration curve (e.g., formed at least inparty by determination of at least one calibration factor).

In yet other embodiments, for example for intermediate concentrationranges where the percentage of active beads may be between about 30% andabout 80%, or between about 40% and about 70%, or between about 60% andabout 80%, or between about 65% and about 75%, or between about 30% andabout 50%, or between about 35% and about 45%, or near 40%, the measureof the concentration of analyte molecules or particles in the fluidsample may be based on an average of the measure of the concentration ofanalyte molecules or particles as determined by a digital analysismethod and as determined by an analog analysis method. That is, at leastone set of data may be analyzed using a digital analysis method and/orone of the analog analysis methods, as described herein. Upondetermination of the AMB (from the digital and/or analog analysis methodand/or average of the two), the AMB may be compared with the calibrationcurve to determine a measure of the concentration of analyte moleculesin the fluid sample.

In some embodiments, in addition to determining a signal indicative ofthe presence/concentration of analyte molecules, at least one backgroundsignal may be determined. In some cases, prior to calculation of an AMBfrom a set of data, a background data set may be subtracted from theanalyzed data set. The background data set may be collected byaddressing the array of locations prior to spatially segregating thetest sample (e.g., analyte molecules that may be immobilized on aplurality of beads) into the locations and/or following spatialseparation but prior to exposure to a plurality of enzymatic substrates(or other precursor labeling agents) to develop the signal.

In some embodiments, in addition to a plurality of capture objects foranalyte capture, a plurality of control objects may also be providedand/or employed. A control object(s) may be useful for a variety ofpurposes including, but not limited to, identification of theorientation of the plurality of locations (e.g., in the case where theplurality of locations is formed as an array of reaction sites, reactionvessels, etc.), to help determine the quality of the assay, and/or tohelp calibrate the detection system (e.g., optical interrogationsystem), as described below. It should be understood, that more than onetype of control object may be present in any assay format (e.g., a firsttype of control object to determine quality of the assay and a secondtype of control object to act as a location marker), or a single type ofcontrol object may have more than one of the above-described functions.

In some cases, the control objects used to identify the orientation ofthe plurality of locations (e.g., reaction vessels, sites, etc.) on anarray (e.g., function as location marker(s) for an array). For example,a control object may be randomly or specifically distributed on anarray, and may provide one or more reference locations for determiningthe orientation/position of the array. Such a feature may be useful whencomparing multiple images of a portion of the array at different timeintervals. That is, the positions of control objects in the array may beused to register the images. In some cases, the control objects may beuse to provide reference locations in embodiments where a plurality ofimages of small overlapping regions are being combined to form a largerimage.

The presence of control objects in an assay may provide informationregarding the quality of the assay. For example, if a location is foundto contain a control object comprising an enzymatic component but nolabeling agent is present (e.g., the product of which would be presentupon exposure of a control object comprising an enzymatic component to aprecursor labeling agent), this gives an indication that some aspect ofthe assay may not be functioning properly. For example, the quality ofthe reagents may be compromised (e.g., concentration of precursorlabeling agent is too low, decomposition of the precursor labelingagent, etc.), and/or perhaps not all of the locations were exposed tothe precursor labeling agent.

In some embodiments, the control objects may be used to calibration thedetection system. For example, the control objects may output an opticalsignal which may be used to calibration an optical detection system. Insome embodiments, the control objects can be characterized and dopedwith a particular characteristic (e.g., fluorescence, color, absorbance,etc.) which can act as a quality control check for the detection systemperformance.

In some cases, the control objects may be used to standardize ornormalize the system to account for variations of the performance and/orcharacteristics of different system components in different assays, overthe course of time, etc. (e.g., detection system, arrays, reagents,etc.) between different portion of an array used in a test, and/orbetween two different arrays. For example, experimental set-up,parameters and/or variations may lead to changes the intensity of asignal (e.g., fluorescence signal) produced from a single array atdifferent time points, or between at least two arrays at simultaneous ordifferent time points. In addition, in a single array, differentportions of the array may produce different background signals. Suchvariations may lead to changes in calibration signals (e.g.,determination of an average bead signal) between arrays, portions of andarray or at multiple times, which can lead to inaccurate determinationsin some cases. Non-limiting examples of parameters that may causevariation include labeling agent concentration, temperature, focus,intensity of detection light, depth and/or size of the locations in anarray, etc. To account for the effects of some or all of suchvariations, in some embodiments, a plurality of control objects may beutilized. In certain instances, such control objects are essentiallyfree of association with analyte molecules or particles. In certainembodiments, less than about 20%, about 10%, about 5%, about 1%, etc. ofthe control objects are associated with analyte molecules or particles.The control objects may be distinguishable from the capture objects(e.g., each may produce a distinguishable signal) and the system may beconfigured such that any analyte molecules associated with a controlobject are not accounted for in the concentration determination of theanalyte molecules. The signals from the control objects may be used tonormalize the interrogation values between different arrays, or in areasof a single array. For example, because the signals from the controlobjects should be approximately equal between arrays and/or about asingle array, the control object signals may be normalized to anappropriate value and the signals of the non-control objects (e.g., thecapture objects associated with an analyte molecule) may be adjustedaccordingly.

As a specific example, in some cases, a group of control objects beingequal to or less than 10% active may be provided to the array. TheĪ_(single) from the capture objects may be determined by equating thedigital AMB (e.g., Equation 4) and analog AMB (e.g., Equation 8). At lowconcentration of analyte molecules in the fluid sample, the percentageof active control objects in is an analog region and AMB_(analog) forthe fluid sample may be calculated using the Ī_(single) determined usingthe control objects (e.g., not using an Ī_(single) calculated usingcapture objects associated with the analyte molecules from the fluidsample). This approach may reduced any imprecision in AMB_(analog)caused by array-to-array, intra-array, and/or day-to-day variation inĪ_(single) as this value is determined using the control objects (e.g.,which may be calibrated with other determinations and/or interrogationsof control objects).

The control objects may be dispersed throughout the assay array oflocations or may be segregated in a set of locations separated from theassay capture objects. For example, a segregated portion of captureobjects may be provided in a region on the assay site separate from theregion containing capture objects, and the value of Ī_(single) for thesesites provides a specific denominator for Equation 8 for this particularportion of an array or this set of arrays. In such cases, the capturebeads do not necessarily need to be distinguishable from the controlobjects since the control objects are spatially separated from thecapture objects.

In some embodiments, an increased dynamic range may be produced orenhanced through use of an imaging camera with a high resolution. Forexample, the above measurements (e.g., given in Table 2) were obtainedusing a 12-bit camera. The “n”-bit characterization of the electronicresolution of a camera shows that 2^(n) quantized analog intensity unitscan be determined. So for a 12-bit camera, 4096 discrete intensityincrements may be distinguished. Thus, a dynamic range of the order of3.6 logs can be achieved typically, and increasing the resolution of thecamera may expand the dynamic range of concentrations which may beaccurately measured in the digital analysis regime, as described herein.

In general, practice of the invention is not particularly limited to anyspecific dynamic ranges or camera types. Instead of or in addition tothe techniques discussed above, other methods/systems may be employed toexpand or further expand dynamic range. For example, detection of agreater quantity of beads (or other capture objects) may expand thedynamic range. In the examples whose results are tabulated in the Tablesabove, about 13% of beads which were exposed to the sample comprisinganalyte molecules were detected. However in other embodiments,increasing the number of reaction vessels (e.g., locations) interrogatedand/or by using cameras with larger fields of view, up to 100% of thebeads which were exposed to the sample could be detected. By detectingan increased number of beads, the dynamic range could be expanded by atleast 1 more log in the digital counting end of the range (e.g., fromabout 4.5 logs to about 5.5 logs, extending the entire range to about7.5 logs). By using more beads, dynamic range can also be extended bylowering the limit of detection (LOD) of the digital read-out analysismethod/system. For example, by increasing the number of beads, thelimiting effects of Poisson noise may be reduced because more eventscould be counted. In certain embodiments, it may be possible, with oneor more of the above described inventive dynamic range extendingtechniques to detect a single analyte molecule per sample. The dynamicrange can also be extended in the higher concentration, analog analysisrange. For example, increasing the electronic resolution of the camera(e.g., linescan with a 24-bit photomultiplier tube or use of advanced16- and 18-bit imaging cameras) may extend the dynamic range of analogmeasurements.

As described below, for embodiments in which a precursor labeling agentis used to facilitate the production of a detectable signal (e.g.,assays where that use enzyme labeled analyte molecules or bindingligands attached to analyte molecules), acquisition of images at shortertime intervals after the analyte molecules have been segregated into theplurality of locations and exposed to a precursor labeling agent that isconverted by to a detected labeling agent (i.e. after shorter incubationtime) may also be able to extend the dynamic range as fewer labelingagent molecules (e.g., converted enzymatic substrate molecules) may bedetected. For example, in the example whose results are tabulated inTable 2, the lowest analog measurement was on 1041 analog counts (seeTable 2). The 12-bit camera used has a dynamic range from 16 counts to65536 counts. In some embodiments, by reducing the incubation time, thedynamic range may be extended by acquiring the measurement at the lowend of the counts (e.g., the lowest analog measurement could potentiallybe made at 16 counts (e.g., instead of 1041 counts)) and thus, the totalanalog dynamic range would be 3.6 logs, equating to a totaldigital+analog dynamic range of about 8.1 logs. A similar effect couldbe achieved by reducing the acquisition time of the image (e.g., howlong the shutter is open for light to fall on the CCD chip). Again, thattime could be minimized to give the lowest possible analog response atthe digital-to-analog switch point and maximize dynamic range. Thesechanges may potentially lead to dynamic range in excess of 9 logs for a12-bit camera in certain embodiments. For example, by implementing thechanges to the digital and analog measurements described here for a12-bit camera a total dynamic range of (5.5 log digital+3.6 log analog)9.1 logs may be achieved. By using 16-, 18-, and 24-bit imaging system,that dynamic range could be extended to 10.3, 10.9, and 12.7, logsrespectively.

In some cases, the digital-to-analog conversion methods described hereinmay include techniques and analysis to account for substrate depletionand/or photobleaching. Substrate depletion may occur in embodimentswhere the assay involves detecting a labeling agent (e.g., fluorescentenzymatic product) which is formed from a precursor labeling agent(e.g., enzymatic substrate) upon exposure to an analyte molecule (orbinding ligand associated with an analyte molecule). It may beadvantageous to account for substrate depletion, for example, inembodiments where a reaction vessel contains more than one analytemolecule (or binding ligand). For example, in a certain experiment,consider that approximately 2.1 million substrate molecules areavailable for turnover in a reaction vessel. In this example, theenzymatic component, beta-galactosidase has a turnover rate of 186 s⁻¹at 100 μM substrate concentration. If there is only one enzymaticcomponent or molecule (or binding ligand) per bead (e.g., present in areaction vessel), that enzymatic component retains 99% of its activityover a duration of a three minute experiment. If, on the other hand,there are on average 50 enzymatic components or molecules per bead (orper reaction vessel, as in a higher concentration sample), approximately43% of the overall activity may be lost of the duration of theexperiment due to substrate depletion. This loss in overall reactionchamber activity can result in a decreased intensity value than would beexpected from a chamber containing ten enzymatic components ormolecules.

To mitigate substrate depletion effects on the accuracy of analogmeasurements, in some embodiments, various techniques may be employed,two examples of which are discussed here. First, the duration of thetime to collect the data set which is analyzed may be reduced. Forexample, fluorescent images at t=0 and t=30 s may be used to calculatethe intensity value instead of a longer duration (e.g., t=0 and t=150s). Performing a measurement over a shorter time period may reduce theamount of substrate that is depleted, and may aid in maintaining alinear rate of fluorescent product formation. Additionally, exposing thereaction chamber containing fluorescent product to excitation lighttwice (e.g., at t=0 and t=30) versus a larger number of times (e.g., att=0, 30, 60, 90, 120, 150 s) may reduce photobleaching effects. Table 3illustrates the reduction in enzyme activity over time due to substratedepletion for an exemplary assay embodiment.

TABLE 3 Reduction in enzyme activity over time # enz/beaded well 100 μMRDG 1 5 10 turnover rate decline over time t (s) 0 505 505 505 30 504498 492 60 502 492 478 90 501 485 464 120 500 478 448 150 498 471 432180 497 464 416 % activity remaining from t = 0 t (s) 0 100% 100% 100%30 100%  99%  97% 60 100%  97%  95% 90  99%  96%  92% 120  99%  95%  89%150  99%  93%  86% 180  98%  92%  82%

For example, FIG. 6 shows a theoretical curve of the amount of RGPconverted into resorufin over the course of an assay measurement from a100 μM RPG solution as a function of the number of enzyme molecules in asealed well containing a bead, taking substrate depletion into account.Assuming that the assay is conducted using a camera with unlimitedsensitivity (e.g., not limited by the number of bits) and imagingstarting immediately after sealing, an assay may remain approximatelylinear to over AMB=50. Specifically, FIG. 6 shows a plot of the numberof resorufin molecules produced during a 150 second experiment as afunction of the number of enzymes on a bead wherein is the theoreticallimit of a modeled where (i) image acquisition begins at t=0,immediately when the seal is made, and before any RGP is converted intoresorufin; and (ii) where image acquisition begins at t=45 second aftersealing.

Another technique which may be employed to mitigate the effects ofsubstrate depletion involves increasing the substrate concentration. TheK_(m) of the enzyme/substrate pair in the above example was about 62 μM.The turnover rate of an enzyme is defined by the substrate concentrationas elucidated by the Michaelis-Menten equation. At substrateconcentrations much greater than the K_(m), the enzyme turnover rate maybegin to plateau. For example, at about 400 μM substrate, the averageenzyme turnover rate is about 707 s⁻¹, while at about 200 μM theturnover rate is about 624 s⁻¹, and at about 100 μM the average turnoveris about 504 s⁻. Reducing the concentration of the substrate in halffrom about 400 μM to about 200 μM results in about 11% reduction inturnover rate, while reducing the concentration of substrate from about200 aM to about 100 μM results in about 20% reduction in turnover rate.Consequently, if substrate depletion occurs during an assay measurementwhile using high substrate concentrations, the depletion may have asmaller effect on the enzyme turnover rate as compared to depletionoccurring when using a lower substrate concentration (e.g., close toK_(m)). Table 4 illustrates an example of the change in depletioneffects as the substrate concentration is altered.

TABLE 4 Depletion effects as substrate concentration is altered 100 μMRDG 200 μM RDG turnover rate # enz/beaded turnover rate # enz/beadeddecline over time well decline over time well t (s) 10 t (s) 10 0 505 0624 30 492 30 618 60 478 60 611 90 464 90 605 120 448 120 597 150 432150 590 180 416 180 582 % activity # enz/beaded % activity # enz/beadedremaining from well remaining from well t (s) 10 t (s) 10 0 100%  0100%  30 97% 30 99% 60 95% 60 98% 90 92% 90 97% 120 89% 120 96% 150 86%150 95% 180 82% 180 93%

To mitigate photobleaching effects on raw fluorescence intensity, insome embodiments, the photobleaching rate of the fluorescent product maybe determined. In the example shown in FIG. 7, the photobleaching ratewas determined by enclosing a solution containing a fluorescent productbeing detected (10 μM resorufin) in the reaction vessels and monitoringthe fluorescence decrease every 15 s with an exposure time of 2000 msover 50 min. An exponential fit of the data yielded a photobleachingrate, k_(ph), of 0.0013 s⁻¹ (see FIG. 7). A unique k_(ph) can bedetermined for the specific optical parameters used in each assay systemset-up and can be used to adjust the data set collected for thephotobleaching effects on raw fluorescence intensities.

The following sections provide additional description, examples andguidance related to various aspects of analytical assay methods/systems,analyte molecules, analyzer systems, etc., that may be used to practicevarious embodiments of the inventive analysis methods/systems describedabove. Additional information may also be found in U.S. patentapplication Ser. No. 12/731,130, entitled “Ultra-Sensitive Detection ofMolecules or Particles using Beads or Other Capture Objects” by Duffy etal., filed Mar. 24, 2010; International Patent Application No.PCT/US11/026,645, entitled “Ultra-Sensitive Detection of Molecules orParticles using Beads or Other Capture Objects” by Duffy et al., filedMar. 1, 2011; U.S. Patent Application No. 20070259448, entitled “Methodsand arrays for target analyte detection and determination of targetanalyte concentration in solution,” by Walt et al., filed Feb. 16, 2007;U.S. Patent Application No. 20070259385, entitled “Methods and arraysfor detecting cells and cellular components in small defined volumes,”by Walt et al., filed Feb. 16, 2007; U.S. Patent Application No.20070259381, entitled “Methods and arrays for target analyte detectionand determination of reaction components that affect a reaction” by Waltet al., filed Feb. 16, 2007; U.S. patent application Ser. No.12/731,135, entitled “Ultra-Sensitive Detection of Molecules using DualDetection Methods” by Duffy et al., filed Mar. 24, 2010; InternationalPatent Application No. PCT/US11/026,657, entitled “Ultra-SensitiveDetection of Molecules using Dual Detection Methods” by Duffy et al.,filed Mar. 1, 2011; International Patent Application No.PCT/US07/019184, entitled “Methods for Determining The Concentration ofan Analyte In Solution” by Walt et al., filed Aug. 20, 2007; andInternational Patent Application No. PCT/US09/005428, entitled“Ultra-Sensitive Detection of Molecules or Enzymes” by Duffy et al.,filed Sep. 9, 2009, herein incorporated by reference.

Methods and Systems for Segregating Analyte Molecules into Arrays ofLocations

In certain embodiments, the assay methods and systems of the presentinvention employ a step of spatially segregating analyte molecules intoa plurality of locations to facilitate detection/quantification, suchthat each location comprises/contains either zero or one or more analytemolecules. Additionally, in some embodiments, the article comprising thelocations is configured in a manner such that each location can beindividually addressed. While exemplary embodiments for spatiallysegregating a plurality of analyte molecules into a plurality oflocations are described herein, numerous other methods may potentiallybe employed.

In some embodiments, an inventive method for determining a measure ofthe concentration of analyte molecules in a fluid sample comprisesdetecting analyte molecules immobilized with respect to a bindingsurface having affinity for at least one type of analyte molecule. Incertain embodiments the binding surface may form (e.g., a surface of awell/reaction vessel on a substrate) or be contained within (e.g., asurface of a capture object, such as a bead, contained within a well)one of a plurality of locations (e.g., a plurality of wells/reactionvessels) on a substrate (e.g., plate, dish, chip, optical fiber end,etc). At least a portion of the locations may be addressed and a measureindicative of the number/percentage of the locations containing at leastone analyte molecule or particle may be made. In some cases, based uponthe number/percentage, a measure of the concentration of analytemolecules or particles in the fluid sample may be determined. Themeasure of the concentration of analyte molecules or particles in thefluid sample may be determined ob a digital analysis method/systemoptionally employing Poisson distribution adjustment and/or based atleast in part on a measured intensity of a signal, as has been describedin detail above.

As described above, in embodiments where the analyte molecules areimmobilized with respect to a plurality of capture objects, thelocations addressed may be locations which contain at least one captureobject (e.g., either associated with or not associated with any analytemolecules), and thus, in these embodiments, the percentage of locationscontaining at least one analyte molecule is also the percentage ofcapture objects associated with at least one analyte molecule (e.g., thepercentage “active” beads). Thus, in some embodiments, a method fordetermining a measure of the concentration of analyte molecules orparticles in a fluid sample comprises exposing a plurality of captureobjects (e.g., beads), each including a binding surface having affinityfor at least one type of analyte molecule or particle (e.g., a pluralityof capture components), to a solution containing or suspected ofcontaining the at least one type of analyte molecules or particles,wherein at least some of the capture objects become associated with atleast one analyte molecule or particle. At least a portion of thecapture objects (e.g., beads) may be spatially segregated into aplurality of locations (e.g., reaction vessels on a surface). At least aportion of the plurality of locations (e.g., in some cases, locationscontaining at least one capture object) may be addressed to determine ameasure indicative of the percentage of locations containing at leastone analyte molecule or particle (e.g., in some cases, the percentage ofcapture objects associated with at least one analyte molecule). Asdescribed above, in some cases, based upon the determined percentage, ameasure of the concentration of analyte molecules or particles in thefluid sample may be determined based at least in part on thenumber/percentage of capture objects containing at least one analytemolecule or particle and/or based at least in part on a measuredintensity of a signal that is indicative of the presence of a pluralityof analyte molecules or particles.

Additionally, in some cases, a system for determining a measure of theconcentration of analyte molecules or particles in a fluid samplecomprises an assay substrate (e.g., plate, dish, slide, chip, opticalfiber face, etc.) comprising a plurality of locations (e.g., reactionvessels) each comprising a binding surface forming (e.g., a plurality ofcapture components) or containing such a surface (e.g., containing abead comprising a plurality of capture components) within suchlocations, wherein at least one binding surface comprises at least oneanalyte molecule or particle immobilized on the binding surface. Thesystem may also comprise at least one detector configured to address atleast a portion of the plurality of locations and able to produce atleast one signal indicative of the presence or absence of an analytemolecule or particle at each location addressed and having the abilityto measure intensity levels varying with the number of analyte moleculesor particles at each location. Additionally, the system may comprise atleast one signal processor configured to determine from the at least onesignal the number/percentage of the addressed locations containing atleast one analyte molecule or particle, and further configured to, basedupon the number/percentage, determine a measure of the concentration ofanalyte molecules or particles in the fluid sample based at least inpart on the number of locations containing at least one analyte moleculeor particle (digital/binary analysis), and/or determine a measure of theconcentration of analyte molecules or particles in the fluid samplebased at least in part on an intensity level of the at least one signalindicative of the presence of a plurality of analyte molecules orparticles, using techniques described previously.

The assay methods and systems provided herein may employ a variety ofdifferent components, steps, and aspects as described herein. Forexample, a method may further comprise determining at least onebackground signal determination (e.g., and further comprisingsubtracting the background signal from other determinations), washsteps, and the like. In some cases, the assays or systems may includethe use of at least one binding ligand, as described herein. In somecases, the measure of the concentration of analyte molecules in a fluidsample is based at least in part on comparison of a measured parameterto a calibration curve. In some instances, the calibration curve isformed at least in part by determination at least one calibrationfactor, as described above.

In some embodiments, the plurality of analyte molecules may be spatiallysegregated into a plurality of locations, wherein the locations comprisea plurality of reaction vessels. The analyte molecules may bepartitioned across the plurality of reaction vessels such that at leastsome of the reaction vessels contain at least one analyte molecule and astatistically significant fraction of the reactions vessels contain noanalyte molecules. A statistically significant fraction of reactionvessels that contain at least one analyte molecule (or no analytemolecules) will typically be able to be reproducibly detected andquantified using a particular system of detection and will typically beabove the background noise (e.g., non-specific binding) that isdetermined when carrying out the assay with a sample that does notcontain any analyte molecules, divided by the total number of locationsaddressed. A “statistically significant fraction” as used herein for thepresent embodiments, may be estimated according to the Equation 10:n>3/√{square root over (n)}  (Eq. 10)wherein n is the number of determined events for a selected category ofevents. That is, a statistically significant fraction occurs when thenumber of events n is greater than three times square root of the numberof events. For example, to determine a statistically significantfraction of the reaction vessels which contain an analyte molecule orparticle, n is the number of reaction vessels which contain an analytemolecule. As another example, to determine a statistically significantfraction of the capture objects associated with a single analytemolecule, n is the number of capture objects associated with a singleanalyte molecule.

In some embodiments, the statistically significant fraction of locationsthat contain at least one analyte molecule (or a single analyte moleculein some cases where the ratio of locations to analyte molecules wouldlead, statistically, to essentially only zero or one analyte moleculecontained in each location) to the total number of locations (or captureobjects) is less than about 1:2, less than about 1:3, less than about1:4, less than about 2:5, less than about 1:5, less than about 1:10,less than about 1:20, less than about 1:100, less than about 1:200, orless than about 1:500. Therefore, in such embodiments, the fraction oflocations (or capture objects) not containing any analyte molecules tothe total number of locations (or capture objects) is at least about1:100, about 1:50, about 1:20, about 1:10, about 1:5, about 1:4, about1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1,about 10:1, about 20:1, about 50:1, about 100:1, or greater.

In some embodiments, as noted previously, the percentage of locationswhich contain at least one analyte molecules is less than about 50%,less than about 40%, less than about 30%, less than about 20%, less thanabout 10%, less than about 5%, less than about 2%, less than about 1%,less than about 0.5%, less than about 0.01%, or less, the total numberof locations (or capture objects). In some embodiments, the percentageof locations which do not contain (or capture object associated with)any analyte molecule is at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, at least about 98%, or greater,the total number of locations (or capture objects).

Methods and techniques for partitioning a plurality of analyte moleculesor particles into a plurality of reaction vessels is described in U.S.patent application Ser. No. 12/731,130, entitled “Ultra-SensitiveDetection of Molecules or Particles using Beads or Other CaptureObjects” by Duffy et al., filed Mar. 24, 2010; International PatentApplication No. PCT/US11/026,645, entitled “Ultra-Sensitive Detection ofMolecules or Particles using Beads or Other Capture Objects” by Duffy etal., filed Mar. 1, 2011; U.S. Patent Application No. 20070259448,entitled “Methods and arrays for target analyte detection anddetermination of target analyte concentration in solution,” by Walt etal., filed Feb. 16, 2007; U.S. Patent Application No. 20070259385,entitled “Methods and arrays for detecting cells and cellular componentsin small defined volumes,” by Walt et al., filed Feb. 16, 2007; U.S.Patent Application No. 20070259381, entitled “Methods and arrays fortarget analyte detection and determination of reaction components thataffect a reaction” by Walt et al., filed Feb. 16, 2007; InternationalPatent Application No. PCT/US07/019184, entitled “Methods forDetermining The Concentration of an Analyte In Solution” by Walt et al.,filed Aug. 20, 2007; and International Patent Application No.PCT/US09/005428, entitled “Ultra-Sensitive Detection of Molecules orEnzymes” by Duffy et al., filed Sep. 9, 2009, herein incorporated byreference.

In some embodiments, the assay methods may comprise the use of aplurality of capture objects. The plurality of capture objects (e.g.,beads) may be configured to capture an analyte molecule or particle. Insome cases, the plurality of capture objects comprises a plurality ofbeads. The beads may or may not be magnetic. At least a portion of thecapture objects may be spatially segregated into a plurality oflocations (e.g., reaction vessels/wells). The plurality of analytemolecules may be exposed to a plurality of types of binding ligandsprior to, concurrent with, or following association of the plurality ofanalyte molecules with respect to the capture components. Various otheraspects of assay methods using such capture components are described incommonly owned U.S. patent application Ser. No. 12/731,130, entitled“Ultra-Sensitive Detection of Molecules or Particles using Beads orOther Capture Objects” by Duffy et al., filed Mar. 24, 2010; andInternational Patent Application No. PCT/US11/026645, entitled“Ultra-Sensitive Detection of Molecules or Particles using Beads orOther Capture Objects” by Duffy et al., filed Mar. 1, 2011, each, hereinincorporated by reference. Specifically, the methods and systemsdescribed herein may be used in combination with and in context with thesingle molecules methods and systems described in the above-referencedapplications. In some cases, the capture objects may be themselvesdetectable (e.g., fluorescence emission), and the beads may be selectedsuch that the detection of the beads does not or does not substantiallyinterfere with the detection of the analyte molecules.

In some embodiments, the analyte molecules may be directly detected orindirectly detected. In the case of direct detection, the analytemolecule may comprise a molecule or moiety that may be directlyinterrogated and/or detected (e.g., a fluorescent entity). In the caseof indirect detection, an additional component is used for determiningthe presence of the analyte molecule. In some cases, the analytemolecules may be composed to a precursor labeling agent (e.g., enzymaticsubstrate) and the enzymatic substrate may be converted to a detectableproduct (e.g., fluorescent molecule) upon exposure to an analytemolecule. In some cases, the plurality analyte molecules may be exposedto at least one additional reaction component prior to, concurrent with,and/or following spatially separating at least some of the analytemolecules into a plurality of locations. In some cases, a plurality ofcapture objects at least some associated with at least one analytemolecule may be exposed to a plurality of binding ligands. In certainembodiments, a binding ligand may be adapted to be directly detected(e.g., the binding ligand comprises a detectable molecule or moiety) ormay be adapted to be indirectly detected (e.g., including a componentthat can convert a precursor labeling agent into a labeling agent), asdiscussed more below. More than one type of binding may be employed inany given assay method, for example, a first type of binding ligand anda second type of binding ligand. In one example, the first type ofbinding ligand is able to associate with a first type of analytemolecule and the second type of binding ligand is able to associate withthe first binding ligand. In another example, both a first type ofbinding ligand and a second type of binding ligand may associate withthe same or different epitopes of a single analyte molecule, asdescribed herein.

Certain binding ligands can comprise an entity that is able tofacilitate detection, either directly or indirectly. A component may beadapted to be directly detected in embodiments where the componentcomprises a measurable property (e.g., a fluorescence emission, a color,etc.). A component may facilitate indirect detection, for example, byconverting a precursor labeling agent into a labeling agent (e.g., anagent that is detected in an assay). A “precursor labeling agent” is anymolecule, particle, or the like, that can be converted to a labelingagent upon exposure to a suitable converting agent (e.g., an enzymaticcomponent comprise in a binding ligand). A “labeling agent” is anymolecule, particle, or the like, that facilitates detection, by actingas the detected entity, using a chosen detection technique.

In some embodiments, at least one binding ligand comprises an enzymaticcomponent. In some embodiments, the analyte molecule may comprise anenzymatic component. The enzymatic component may convert a precursorlabeling agent (e.g., an enzymatic substrate) into a labeling agent(e.g., a detectable product). A measure of the concentration of analytemolecules in the fluid sample can then be determined based at least inpart by determining the number of locations containing a labeling agent(e.g., by relating the number of locations containing a labeling agentto the number of locations containing an analyte molecule (or number ofcapture objects associated with at least one analyte molecule to totalnumber of capture objects)). Non-limiting examples of enzymes orenzymatic components include horseradish peroxidase, beta-galactosidase,and alkaline phosphatase. Other non-limiting examples of systems ormethods for detection include embodiments where nucleic acid precursorsare replicated into multiple copies or converted to a nucleic acid thatcan be detected readily, such as the polymerase chain reaction (PCR),rolling circle amplification (RCA), ligation, Loop-Mediated IsothermalAmplification (LAMP), etc. Such systems and methods will be known tothose of ordinary skill in the art, for example, as described in “DNAAmplification: Current Technologies and Applications,” Vadim Demidov etal., 2004.

As an example of an assay method which comprises the use of a precursorlabeling agent, as shown in FIG. 8, substrate 100 comprising a pluralityof locations is provided, wherein the locations comprise reactionvessels. In reaction vessel 101 (e.g., location), analyte molecule 102is immobilized with respect to bead 103 (e.g., capture object). Bindingligand 104 is associated with analyte molecule 102. Binding ligand 104comprises an enzymatic component (not shown). Precursor labeling agent106 is converted to labeling agent 108 (upon exposure to the enzymaticcomponent). Labeling agent 108 is detected using methods describedherein. In contrast, reaction vessel 111 contains analyte molecule 112immobilized with respect to bead 110. In this reaction vessel, analytemolecule 112 is not associated with a binding ligand comprising anenzymatic component. Therefore, precursor labeling agent 114 is notconverted to a labeling agent in the reaction vessel. Thus this reactionvessel would give a different signal as compared to reaction vessel 101where the precursor labeling agent was converted to a labeling agent. Insome cases, there may also be reaction vessels which contain a bead notassociated with an analyte molecule, for example, reaction vessel 121contains bead 116. Additionally, some of the reaction vessels may notcomprise any bead, for example, reaction vessel 123. Reaction vessels121 and 123 may give different signals as compared to reaction vessel101 as there would be no labeling agent present. However, reactionvessels 121 and 123 may contain precursor labeling agent 117. More thanone precursor labeling agent may be present in any given reactionvessel.

In certain embodiments, solubilized, or suspended precursor labelingagents may be employed, wherein the precursor labeling agents areconverted to labeling agents which are insoluble in the liquid and/orwhich become immobilized within/near the location (e.g., within thereaction vessel in which the labeling agent is formed). Such precursorlabeling agents and labeling agents and their use is described incommonly owned U.S. patent application Ser. No. 12/236,484, filed Sep.23, 2008, entitled “High Sensitivity Determination of the Concentrationof Analyte molecules in a Fluid Sample,” by Duffy et al., incorporatedherein by reference.

In some embodiments, techniques may be used to prevent or reducedissociation of an analyte molecule from a capture component and/orcapture object, and/or to prevent or reduce dissociation of a bindingligand from an analyte molecule and/or another binding ligand. As willbe known to those of ordinary skill in the art, some reversible affinityinteractions between selected analyte molecules, capture components,and/or binding ligands (e.g., between an antibody and an antigen) aregoverned by thermodynamics. Accordingly, at some point during certainassay methods, some dissociation may occur between an analyte moleculeand a capture component and/or a binding ligand, and/or between abinding ligand and an analyte molecule and/or another binding ligand.This may result in a reduced number of analyte molecules (e.g.,immunocomplexes) being detected than are actually present. Thedissociation constant of a particular pair of components (e.g.,antibody-antigen pair), washing and/or fluid exposure, time betweenexposure and interrogation, and/or other factors, may affect the degreeto which a dissociation event alters determination of analyte moleculesand/or particles. Accordingly, certain techniques may be used to reducethe effects of dissociation processes.

In a first embodiment, dissociation may be reduced or eliminated byremoving fluids from the assay locations (e.g., wells) following spatialsegregation of a plurality of analyte molecules (e.g., associated with acapture object via a capture component and/or associated with at leastone binding ligand) into a plurality of such locations. That is, all orsubstantially all of the fluid surrounding or substantially contained inor at the locations may be removed. For example, the fluid may beremoved by air and/or vacuum drying. Removal of the fluid may reduce oreliminate dissociation. Immediately prior to interrogation of thelocations, a fluid may be added to the locations thereby rehydrating thecomplexes to facilitate interrogation using a detector.

In a second embodiment, dissociation may be reduced or eliminated bycrosslinking an analyte molecule with a capture component, and/orcrosslinking a binding ligand with an analyte molecule and/or a secondbinding ligand. For example, an analyte molecule comprising an antigenmay be crosslinked with a binding ligand and/or capture componentcomprising an antibody. Crosslinking methods and techniques that may beemployed are known to those of ordinary skill in the art.

In some embodiments, a plurality of locations may be addressed and/or aplurality of capture objects and/or species/molecules/particles ofinterest may be detected substantially simultaneously. “Substantiallysimultaneously” when used in this context, refers toaddressing/detection of the locations/captureobjects/species/molecules/particles of interest at approximately thesame time such that the time periods during which at least twolocations/capture objects/species/molecules/particles of interest areaddressed/detected overlap, as opposed to being sequentiallyaddressed/detected, where they would not. Simultaneousaddressing/detection can be accomplished by using various techniques,including optical techniques (e.g., CCD detector). Spatially segregatingcapture objects/species/molecules/particles into a plurality ofdiscrete, resolvable locations, according to some embodimentsfacilitates substantially simultaneous detection by allowing multiplelocations to be addressed substantially simultaneously. For example, forembodiments where individual species/molecules/particles are associatedwith capture objects that are spatially segregated with respect to theother capture objects into a plurality of discrete, separatelyresolvable locations during detection, substantially simultaneouslyaddressing the plurality of discrete, separately resolvable locationspermits individual capture objects, and thus individualspecies/molecules/particles (e.g., analyte molecules) to be resolved.For example, in certain embodiments, individual molecules/particles of aplurality of molecules/particles are partitioned across a plurality ofreaction vessels such that each reaction vessel contains zero or onlyone species/molecule/particle. In some cases, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, atleast about 99.5% of all species/molecules/particles are spatiallyseparated with respect to other species/molecules/particles duringdetection. A plurality of species/molecules/particles may be detectedsubstantially simultaneously within a time period of less than about 1second, less than about 500 milliseconds, less than about 100milliseconds, less than about 50 milliseconds, less than about 10milliseconds, less than about 1 millisecond, less than about 500microseconds, less than about 100 microseconds, less than about 50microseconds, less than about 10 microseconds, less than about 1microsecond, less than about 0.5 microseconds, less than about 0.1microseconds, or less than about 0.01 microseconds, less than about0.001 microseconds, or less. In some embodiments, the plurality ofspecies/molecules/particles may be detected substantially simultaneouslywithin a time period of between about 100 microseconds and about 0.001microseconds, between about 10 microseconds and about 0.01 microseconds,or less.

In some embodiments, the locations are optically interrogated. Thelocations exhibiting changes in their optical signature may beidentified by a conventional optical train and optical detection system.Depending on the detected species (e.g., type of fluorescence entity,etc.) and the operative wavelengths, optical filters designed for aparticular wavelength may be employed for optical interrogation of thelocations. In embodiments where optical interrogation is used, thesystem may comprise more than one light source and/or a plurality offilters to adjust the wavelength and/or intensity of the light source.In some embodiments, the optical signal from a plurality of locations iscaptured using a CCD camera.

Other non-limiting examples of camera imaging types that can be used tocapture images include charge injection devices (CIDs), complimentarymetal oxide semiconductors (CMOSs) devices, scientific CMOS (sCMOS)devices, and time delay integration (TDI) devices, as will be known tothose of ordinary skill in the art. The camera may be obtained from acommercial source. CIDs are solid state, two dimensional multi pixelimaging devices similar to CCDS, but differ in how the image is capturedand read. For examples of CCDs, see U.S. Pat. Nos. 3,521,244 and4,016,550. CMOS devices are also two dimensional, solid state imagingdevices but differ from standard CCD arrays in how the charge iscollected and read out. The pixels are built into a semiconductortechnology platform that manufactures CMOS transistors thus allowing asignificant gain in signal from substantial readout electronics andsignificant correction electronics built onto the device. For example,see U.S. Pat. No. 588,383). sCMOS devices comprise CMOS imagingtechnology with certain technological improvements that allows excellentsensitivity and dynamic range. TDI devices employs a CCD device whichallows columns of pixels to be shifted into and adjacent column andallowed to continue gathering light. This type of device is typicallyused in such a manner that the shifting of the column of pixels issynchronous with the motion of the image being gathered such that amoving image can be integrated for a significant amount of time and isnot blurred by the relative motion of the image on the camera. In someembodiments, a scanning mirror system coupled with a photodiode orphotomultiplier tube (PMT) could be used to for imaging.

In one embodiment, the plurality of locations is formed directly as aplurality of reaction vessels in an end of a fiber optic bundle.According to one embodiment, the array of reaction vessels for thepresent invention can be used in conjunction with an optical detectionsystem such as the system described in U.S. Publication No. 20030027126.For example, according to one embodiment, the array of reaction vesselsof the present invention is formed in one end of a fiber optic assemblycomprising a fiber optic bundle constructed of clad fibers so that lightdoes not mix between fibers.

FIGS. 9A and 9B show non-limiting examples of a system of the presentinvention according to some embodiments. The system comprises a lightsource 452, excitation filter 454, dichromatic mirror 458, emissionfilter 460, objective 470, and array 472. Light 453 given off from lightsource 452 is passed through excitation filter 454. The light reflectsoff dichromatic mirror 458, passes through objective 470 and shines onarray 472. In some cases, stray light 464 may be reduced by a straylight reducing function 468, such as an iris or aperture. Light 471emitted from the array passes through objective 470 and emission filter460. Light 462 is observed. The system may comprise additionalcomponents (e.g., additional filters, mirrors, magnification devices,etc.) as needed for particular applications, as would be understood bythose of ordinary skill in the art.

The system shown in FIG. 9A may additionally comprise components whichaid in the determination of the number of reaction vessels which containa capture object (e.g., using white light). Alternatively, theadditional components may be used to determine the total number oflocations and/or provide spatially information regarding the position ofthe locations (e.g., containing or not containing a capture object),which may help corroborate signals observed under different lightregimes (e.g., fluorescence, white light) corresponding with theposition of a location (e.g., a mask may be created).

In FIGS. 9A and 9B, excitation light is emitted from source 452 andcollimated into a beam 453. The excitation filter 454 may be configuredto transmit only the wavelength band that excites the fluorophore (e.g.,575 nm+/−10 nm for resorufin). The excitation light is reflecteddownward by the dichroic filter 458 and excites the substrate 472containing the sample through the objective lens 470. The image light iscollected by the objective lens 470, collimated into a beam 471 andtransmitted through the dichroic filter 458. Only the image lightcorresponding to the fluorescence wavelength band (e.g., 670 nm+/−30 nmfor resorufin) is transmitted through the emission filter 460. Theremaining collimated beam 462 contains only the emitted fluorescencewavelengths which will subsequently be imaged through the camera system.

The same system may be used to determine the positioning of thelocations containing sample (e.g., reaction vessels). The arraycomprising the reaction vessels containing capture objects may beilluminated with a “bright field” white light illumination. The arraymay be illuminated (e.g., using light source 475 shown in FIG. 9A) bydirecting a pseudo-collimated white light (e.g., white light LED) ontothe array surface from an angle (e.g., θ₁ in FIG. 9A may be about 20degrees, about 25 degrees, about 30 degrees, about 35 degrees, about 40degrees, or greater) just outside the numerical aperture of thecollection objective. Light that hits the surface of the array 472(e.g., light 476) is reflected (and scattered) off the surface,collimated 471, and collected by the objective lens (470). Thecollimated beam is subsequently imaged through the camera system.

The same system may also be used to determine which locations contain acapture object (e.g., bead). Any particular bead may or may not beassociated with an analyte molecule and/or binding ligand. The array maybe illuminated (e.g., using light source 473 as shown in FIG. 9A) with a“dark field” white light illumination. The array may be illuminated byaiming a pseudo-collimated white light (e.g., white light LED 473) ontothe array surface from an angle (e.g., θ₂ in FIG. 9A is about 65degrees, about 70 degrees, about 75 degrees, about 80 degrees, about 85degrees) substantially outside the numerical aperture of the collectionobjective. Light that hits the surface of the array 472 (e.g., light474) is reflected (and scattered) off the surface, collimated 471, andcollected by the objective lens 470. The collimated beam is subsequentlyimaged by the camera system.

In some embodiments, an optical detection system may be employed, forexample, as described in U.S. Publication No. 2003/0027126. In anexemplary system, light returning from an array of reaction vesselsformed at the distal end of a fiber optic bundle is altered via use of amagnification changer to enable adjustment of the image size of thefiber's proximal or distal end. The magnified image is then shutteredand filtered by a shutter wheel. The image is then captured by chargecoupled device (CCD) camera. A computer may be provided that includesand executes imaging processing software to process the information fromthe CCD camera and also optionally may be configured to control shutterand filter wheels. As depicted in U.S. Publication No. 20030027126, theproximal end of the bundle is received by a z-translation stage and x-ymicropositioner.

For example, FIG. 10 shows a schematic block diagram of a systememploying a fiber optic assembly 400 with an optical detection system.The fiber optic assembly 400 that comprises a fiber optic bundle orarray 402 that is constructed from clad fibers so that light does notmix between fibers. An array of reaction vessels 401 is formedat/attached to the bundle's distal end 412, with the proximal end 414being operatively connected with a z-translation stage 416 and x-ymicropositioner 418. These two components act in concert to properlyposition the proximal end 414 of the bundle 402 for a microscopeobjective lens 420. Light collected by the objective lens 420 is passedto a reflected light fluorescence attachment with three pointer cubeslider 422. The attachment 422 allows directs light from a 75 watt Xelamp 424 through the objective lens 420 to be coupled into the fiberbundle 402. The light from source 424 is condensed by condensing lens426, then filtered and/or shuttered by filter and shutter wheel 428, andsubsequently passes through a ND filter slide 430. Light returning fromthe distal end 412 of the bundle 402 passes through the attachment 422to a magnification changer 432 which enables adjustment of the imagesize of the fiber's proximal or distal end. Light passing through themagnification changer 432 is then shuttered and filtered by a secondwheel 434. The light is collected by a charge coupled device (CCD)camera 436. A computer 438 executes imaging processing software toprocess the information from the CCD camera 436 and also optionallycontrols other components of the system, including but not limited tothe first and second shutter and filter wheels 428, 434. An array ofreaction vessels used to practice some embodiments of the presentinvention may be integral with or attached to the distal end of thefiber optic bundle using a variety of compatible processes. In somecases, microwells are formed at the center of each individual fiber ofthe fiber optic bundle and the microwells may or may not be sealed. Eachoptical fiber of the fiber optic bundle may convey light from the singlemicrowell formed at the center of the fiber's distal end. This featureenables the interrogation of the optical signature of individualreaction vessels to identify reactions/contents in each microwell.Consequently, by collecting the image of the end of the bundle with theCCD array, the optical signatures of the reaction vessels may beindividually interrogated and/or imaged substantially simultaneously.

The plurality of locations may be formed using any suitable technique.In some embodiments, the plurality of locations comprises a plurality ofreaction vessels/wells on a substrate. The reactions vessels, in certainembodiments, may be configured to receive and contain only a singlecapture object.

In some embodiments of the present invention, the plurality of reactionvessels may be sealed (e.g., after the introduction of the analytemolecules, binding ligands, and/or precursor labeling agent), forexample, through the mating of the second substrate and a sealingcomponent. The sealing of the reaction vessels may be such that thecontents of each reaction vessel cannot escape the reaction vesselduring the remainder of the assay. In some cases, the reaction vesselsmay be sealed after the addition of the analyte molecules and,optionally, at least one type of precursor labeling agent to facilitatedetection of the analyte molecules. For embodiments employing precursorlabeling agents, by sealing the contents in some or each reactionvessel, a reaction to produce the detectable labeling agents can proceedwithin the sealed reaction vessels, thereby producing a detectableamount of labeling agents that is retained in the reaction vessel fordetection purposes.

The plurality of locations comprising a plurality of reaction vesselsmay be formed using a variety of methods and/or materials. In somecases, the plurality of reaction vessels is formed as an array ofdepressions on a first surface. In other cases, however, the pluralityof reaction vessels may be formed by mating a sealing componentcomprising a plurality of depressions with a substrate that may eitherhave a featureless surface or include depressions aligned with those onthe sealing component. Any of the device components, for example, thesubstrate or sealing component, may be fabricated from a compliantmaterial, e.g., an elastomeric polymer material, to aid in sealing. Thesurfaces may be or made to be hydrophobic or contain hydrophobic regionsto minimize leakage of aqueous samples from the microwells.

In some cases, the sealing component may be capable of contacting theexterior surface of an array of microwells (e.g., the cladding of afiber optic bundle as described in more detail below) such that eachreaction vessel becomes sealed or isolated such that the contents ofeach reaction vessel cannot escape the reaction vessel. According to oneembodiment, the sealing component may be a silicone elastomer gasketthat may be placed against an array of microwells with application ofsubstantially uniform pressure across the entire substrate. In somecases, the reaction vessels may be sealed after the addition of theplurality of capture objects used for analyte capture and, optionally,any precursor labeling agent molecule that may be used to facilitatedetection of the analyte molecule.

A non-limiting example of the formation of a plurality of reactionvessels containing assay solution on/in a substrate is depicted in FIG.11. FIG. 11, panel (A) shows a surface comprising a plurality ofmicrowells 139, which have been exposed to an assay solution 141 (e.g.,a solution containing the analyte molecules), and a sealing component143. Sealing component 143 in this example comprises a substantiallyplanar bottom surface. Mating of substrate 139 with sealing component143 forms a plurality of sealed reaction vessels 145. The areas betweenthe reaction vessels 148 may be modified to aid in the formation of atight seal between the reaction vessels.

A second embodiment is shown in FIG. 11, panel (B), in which sealingcomponent 162 comprising a plurality of microwells 163 is mated with asubstantially planar surface 158 which has been exposed to assaysolution 162, thereby forming a plurality of reaction vessels 164.

In a third embodiment, as shown in FIG. 11, panel (C), substrate surface166 comprising a plurality of microwells 167 is mated with sealingcomponent 170 also comprising a plurality of microwells 171. In thisembodiment, the microwells in the substrate and the microwells in thesealing components are substantially aligned so each reaction vessel 172formed comprises a portion of the microwell from the sealing componentand a portion of a microwell from the substrate. In FIG. 11, panel (D),the microwells are not aligned such that each reaction vessel compriseseither a microwell from the sealing component 173 or a microwell fromthe substrate 175.

The sealing component may be essentially the same size as the substrateor may be different in size. In some cases, the sealing component isapproximately the same size as the substrate and mates withsubstantially the entire surface of the substrate. In other cases, asdepicted in FIG. 11, panel (E), the sealing component 176 is smallerthan the substrate 174 and the sealing component only mates with aportion 178 of the substrate. In yet another embodiment, as depicted inFIG. 11, panel (F), the sealing component 182 is larger than thesubstrate 180, and only a portion 184 of the sealing component mateswith the substrate 180.

In some embodiments, the reaction vessels may all have approximately thesame volume. In other embodiments, the reaction vessels may havediffering volumes. The volume of each individual reaction vessel may beselected to be appropriate to facilitate any particular assay protocol.For example, in one set of embodiments where it is desirable to limitthe number of capture objects used for analyte capture contained in eachvessel to a small number, the volume of the reaction vessels may rangefrom attoliters or smaller to nanoliters or larger depending upon thenature of the capture objects, the detection technique and equipmentemployed, the number and density of the wells on the substrate and theexpected concentration of capture objects in the fluid applied to thesubstrate containing the wells. In one embodiment, the size of thereaction vessel may be selected such only a single capture object usedfor analyte capture can be fully contained within the reaction vessel(see, for example, U.S. patent application Ser. No. 12/731,130, entitled“Ultra-Sensitive Detection of Molecules or Particles using Beads orOther Capture Objects” by Duffy, et al., filed Mar. 24, 2010; orInternational Patent Application No. PCT/US11/026,645, entitled“Ultra-Sensitive Detection of Molecules or Particles using Beads orOther Capture Objects” by Duffy, et al., filed Mar. 1, 2011, hereinincorporated by reference).

In accordance with one embodiment of the present invention, the reactionvessels may have a volume between about 1 femtoliter and about 1picoliter, between about 1 femtoliters and about 100 femtoliters,between about 10 attoliters and about 100 picoliters, between about 1picoliter and about 100 picoliters, between about 1 femtoliter and about1 picoliter, or between about 30 femtoliters and about 60 femtoliters.In some cases, the reaction vessels have a volume of less than about 1picoliter, less than about 500 femtoliters, less than about 100femtoliters, less than about 50 femtoliters, or less than about 1femtoliter. In some cases, the reaction vessels have a volume of about10 femtoliters, about 20 femtoliters, about 30 femtoliters, about 40femtoliters, about 50 femtoliters, about 60 femtoliters, about 70femtoliters, about 80 femtoliters, about 90 femtoliters, or about 100femtoliters.

The total number of locations and/or density of the locations employedin an assay (e.g., the number/density of reaction vessels in an array)can depend on the composition and end use of the array. For example, thenumber of reaction vessels employed may depend on the number of types ofanalyte molecule and/or binding ligand employed, the suspectedconcentration range of the assay, the method of detection, the size ofthe capture objects, the type of detection entity (e.g., free labelingagent in solution, precipitating labeling agent, etc.). Arrayscontaining from about 2 to many billions of reaction vessels (or totalnumber of reaction vessels) can be made by utilizing a variety oftechniques and materials. Increasing the number of reaction vessels inthe array can be used to increase the dynamic range of an assay or toallow multiple samples or multiple types of analyte molecules to beassayed in parallel. The array may comprise between one thousand and onemillion reaction vessels per sample to be analyzed. In some cases, thearray comprises greater than one million reaction vessels. In someembodiments, the array comprises between about 1,000 and about 50,000,between about 1,000 and about 1,000,000, between about 1,000 and about10,000, between about 10,000 and about 100,000, between about 100,000and about 1,000,000, between about 100,000 and about 500,000, betweenabout 1,000 and about 100,000, between about 50,000 and about 100,000,between about 20,000 and about 80,000, between about 30,000 and about70,000, between about 40,000 and about 60,000 reaction vessels. In someembodiments, the array comprises about 10,000, about 20,000, about50,000, about 100,000, about 150,000, about 200,000, about 300,000,about 500,000, about 1,000,000, or more, reaction vessels.

The array of reaction vessels may be arranged on a substantially planarsurface or in a non-planar three-dimensional arrangement. The reactionvessels may be arrayed in a regular pattern or may be randomlydistributed. In a specific embodiment, the array is a regular pattern ofsites on a substantially planar surface permitting the sites to beaddressed in the X-Y coordinate plane.

In some embodiments, the reaction vessels are formed in a solidmaterial. As will be appreciated by those in the art, the number ofpotentially suitable materials in which the reaction vessels can beformed is very large, and includes, but is not limited to, glass(including modified and/or functionalized glass), plastics (includingacrylics, polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, cyclic olefincopolymer (COC), cyclic olefin polymer (COP), Teflon®, polysaccharides,nylon or nitrocellulose, etc.), elastomers (such as poly(dimethylsiloxane) and poly urethanes), composite materials, ceramics, silica orsilica-based materials (including silicon and modified silicon), carbon,metals, optical fiber bundles, or the like. In general, the substratematerial may be selected to allow for optical detection withoutappreciable autofluorescence. In certain embodiments, the reactionvessels may be formed in a flexible material.

A reaction vessel in a surface (e.g., substrate or sealing component)may be formed using a variety of techniques known in the art, including,but not limited to, photolithography, stamping techniques, moldingtechniques, etching techniques, or the like. As will be appreciated bythose of the ordinary skill in the art, the technique used can depend onthe composition and shape of the supporting material and the size andnumber of reaction vessels.

In a particular embodiment, an array of reaction vessels is formed bycreating microwells on one end of a fiber optic bundle and utilizing aplanar compliant surface as a sealing component. In certain suchembodiments, an array of reaction vessels in the end of a fiber opticbundle may be formed as follows. First, an array of microwells is etchedinto the end of a polished fiber optic bundle. Techniques and materialsfor forming and etching a fiber optic bundle are known to those ofordinary skill in the art. For example, the diameter of the opticalfibers, the presence, size and composition of core and cladding regionsof the fiber, and the depth and specificity of the etch may be varied bythe etching technique chosen so that microwells of the desired volumemay be formed. In certain embodiments, the etching process createsmicrowells by preferentially etching the core material of the individualglass fibers in the bundle such that each well is approximately alignedwith a single fiber and isolated from adjacent wells by the claddingmaterial. Potential advantages of the fiber optic array format is thatit can produce thousands to millions of reaction vessels withoutcomplicated microfabrication procedures and that it can provide theability to observe and optically address many reaction vesselssimultaneously.

Each microwell may be aligned with an optical fiber in the bundle sothat the fiber optic bundle can carry both excitation and emission lightto and from the wells, enabling remote interrogation of the wellcontents. Further, an array of optical fibers may provide the capabilityfor simultaneous or non-simultaneous excitation of molecules in adjacentvessels, without signal “cross-talk” between fibers. That is, excitationlight transmitted in one fiber does not escape to a neighboring fiber.

Alternatively, the equivalent structures of a plurality of reactionvessels may be fabricated using other methods and materials that do notutilize the ends of an optical fiber bundle as a substrate. For example,the array may be a spotted, printed or photolithographically fabricatedsubstrate produced by techniques known in the art; see for exampleWO95/25116; WO95/35505; PCT US98/09163; U.S. Pat. Nos. 5,700,637,5,807,522, 5,445,934, 6,406,845, and 6,482,593. In some cases, the arraymay be produced using molding, embossing, and/or etching techniques aswill be known to those of ordinary skill in the art.

In certain embodiments, the present invention provides a system equippedwith a mechanical platform that applies a sealing component to asubstrate. The platform may be positioned beneath a stage on the system.After the chosen reaction components have been added to an array ofreaction vessels, the sealing component may be mated with the array. Forexample, the sealing component may be sandwiched between a flat surface(such as, for example, a microscope slide) and the array of reactionvessels using uniform pressure applied by the mechanical platform.

A non-limiting embodiment is illustrated in FIG. 12A. A sealingcomponent 300 is placed on top of mechanical platform 302. The assaysolution 304 is placed on top of the sealing component 300. Themechanical platform is moved upwards towards the array 306 (e.g., fiberoptic array) such that uniform pressure is applied. As shown in FIG.12B, the sealing component 300 forms a tight seal with the array 306. Inother instances, varying pressure may be applied to the sealingcomponent to form a tight seal between the sealing component and thearray. The system may also comprise additional components 312 that maybe utilized to analyze the array (e.g., microscope, computer, etc.) asdiscussed more herein.

In some embodiments, the plurality of locations may not comprise aplurality of reaction vessels/wells. For example, in embodiments wherecapture objects are employed, a patterned substantially planar surfacemay be employed and the patterned areas form a plurality of locations.In some cases, the patterned areas may comprise substantiallyhydrophilic surfaces which are substantially surrounded by substantiallyhydrophobic surfaces. In certain embodiments, a plurality of captureobjects (e.g., beads) may be substantially surrounded by a substantiallyhydrophilic medium (e.g., comprising water), and the beads may beexposed to the patterned surface such that the beads associate in thepatterned areas (e.g., the hydrophilic locations on the surface),thereby spatially segregating the plurality of beads. For example, inone such embodiment, a substrate may be or include a gel or othermaterial able to provide a sufficient barrier to mass transport (e.g.,convective and/or diffusional barrier) to prevent capture objects usedfor analyte capture and/or precursor labeling agent and/or labelingagent from moving from one location on or in the material to anotherlocation so as to cause interference or cross-talk between spatiallocations containing different capture objects during the time framerequired to address the locations and complete the assay. For example,in one embodiment, a plurality of capture objects is spatially separatedby dispersing the capture objects on and/or in a hydrogel material. Insome cases, a precursor labeling agent may be already present in thehydrogel, thereby facilitating development of a local concentration ofthe labeling agent (e.g., upon exposure to a binding ligand or analytemolecule carrying an enzymatic component). As still yet anotherembodiment, the capture objects may be confined in one or morecapillaries. In some cases, the plurality of capture objects may beabsorbed or localized on a porous or fibrous substrate, for example,filter paper. In some embodiments, the capture objects may be spatiallysegregated on a uniform surface (e.g., a planar surface), and thecapture objects may be detected using precursor labeling agents whichare converted to substantially insoluble or precipitating labelingagents that remain localized at or near the location of where thecorresponding capture object is localized. The use of such substantiallyinsoluble or precipitating labeling agents is described herein. In somecases, single analyte molecules may be spatially segregated into aplurality of droplets. That is, single analyte molecules may besubstantially contained in a droplet containing a first fluid. Thedroplet may be substantially surrounded by a second fluid, wherein thesecond fluid is substantially immiscible with the first fluid.

In some embodiments, during the assay, at least one washing step may becarried out. In certain embodiments, the wash solution is selected sothat it does not cause appreciable change to the configuration of thecapture objects and/or analyte molecules and/or does not disrupt anyspecific binding interaction between at least two components of theassay (e.g., a capture component and an analyte molecule). In othercases, the wash solution may be a solution that is selected tochemically interact with one or more assay components. As will beunderstood by those of ordinary skill in the art, a wash step may beperformed at any appropriate time point during the inventive methods.For example, a plurality of capture objects may be washed after exposingthe capture objects to one or more solutions comprising analytemolecules, binding ligands, precursor labeling agents, or the like. Asanother example, following immobilization of the analyte molecules withrespect to a plurality of capture objects, the plurality of captureobjects may be subjected to a washing step thereby removing any analytemolecules not specifically immobilized with respect to a capture object.

Analyzer Systems

The invention also involves a system for determining a measure of theconcentration of analyte molecules or particles in a fluid sampleconfigured to perform at least some of the assay steps and/orsignal/data processing steps described above.

For example in certain embodiments, the invention involves a system fordetermining a measure of the concentration of analyte molecules orparticles in a fluid sample, comprising an assay substrate comprising aplurality of locations each comprising a binding surface forming orcontained within such locations, wherein at least one binding surfacecomprises at least one analyte molecule or particle immobilized on thebinding surface, at least one detector configured to address a pluralityof the locations and able to produce at least one signal indicative ofthe presence or absence of an analyte molecule or particle at eachlocation addressed and having an intensity varying with the number ofanalyte molecules or particles at each location, and at least one signalprocessor configured to determine from the at least one signal thepercentage of the locations containing at least one analyte molecule orparticle, and further configured to, based upon the percentage, eitherdetermine a measure of the concentration of analyte molecules orparticles in the fluid sample based at least in part on the number oflocations containing at least one analyte molecule or particle, ordetermine a measure of the concentration of analyte molecules orparticles in the fluid sample based at least in part on an intensitylevel of the at least one signal indicative of the presence of aplurality of analyte molecules or particles.

In certain such embodiments, the signal processor may comprise or be apart of the computer 438 illustrated in FIG. 10. The signalprocessor/computer can be part of or coupled in operative associationwith the remaining components of the system, and, in some embodiments,configured and/or programmed to control and adjust operationalparameters of the system as well as analyze and calculate values, asdescribed above. In some embodiments, the signal processor/computer cansend and receive control signals to set and/or control operatingparameters of the other components of the system. In other embodiments,the signal processor/computer can be separate from and/or remotelylocated with respect to other system components and may be configured toreceive data from one or more other components of the system viaindirect and/or portable means, such as via portable electronic datastorage devices, such as magnetic disks, or via communication over acomputer network, such as the Internet or a local intranet.

The signal processor/computer may include several known components andcircuitry, including a processing unit (i.e., processor), a memorysystem, input and output devices and interfaces (e.g., aninterconnection mechanism), as well as other components, such astransport circuitry (e.g., one or more busses), a video and audio datainput/output (I/O) subsystem, special-purpose hardware, as well as othercomponents and circuitry, as described below in more detail. Further,the signal processor/computer may be a multi-processor computer systemor may include multiple computers connected over a computer network.

The signal processor/computer may include a processor, for example, acommercially available processor such as one of the series x86, Celeronand Pentium processors, available from Intel, similar devices from AMDand Cyrix, the 680X0 series microprocessors available from Motorola, andthe PowerPC microprocessor from IBM. Many other processors areavailable, and the computer system is not limited to a particularprocessor.

A processor typically executes a program called an operating system, ofwhich Windows 7, Windows Vista, WindowsNT, Windows95 or 98, UNIX, Linux,DOS, VMS, MacOS and OS8 are examples, which controls the execution ofother computer programs and provides scheduling, debugging, input/outputcontrol, accounting, compilation, storage assignment, data managementand memory management, communication control and related services. Theprocessor and operating system together define a computer platform forwhich application programs in high-level programming languages arewritten. The signal processor/computer is not limited to a particularcomputer platform.

The signal processor/computer may include a memory system, whichtypically includes a computer readable and writeable non-volatilerecording medium, of which a magnetic disk, optical disk, a flash memoryand tape are examples. Such a recording medium may be removable, forexample, a floppy disk, read/write CD or memory stick, or may bepermanent, for example, a hard drive.

Such a recording medium stores signals, typically in binary form (i.e.,a form interpreted as a sequence of one and zeros). A disk (e.g.,magnetic or optical) has a number of tracks, on which such signals maybe stored, typically in binary form, i.e., a form interpreted as asequence of ones and zeros. Such signals may define a software program,e.g., an application program, to be executed by the microprocessor, orinformation to be processed by the application program.

The memory system of the signal processor/computer also may include anintegrated circuit memory element, which typically is a volatile, randomaccess memory such as a dynamic random access memory (DRAM) or staticmemory (SRAM). Typically, in operation, the processor causes programsand data to be read from the non-volatile recording medium into theintegrated circuit memory element, which typically allows for fasteraccess to the program instructions and data by the processor than doesthe non-volatile recording medium.

The processor generally manipulates the data within the integratedcircuit memory element in accordance with the program instructions andthen copies the manipulated data to the non-volatile recording mediumafter processing is completed. A variety of mechanisms are known formanaging data movement between the non-volatile recording medium and theintegrated circuit memory element, and the signal processor/computerthat implements the methods, steps, systems and system elementsdescribed above is not limited thereto. The signal processor/computer isnot limited to a particular memory system.

At least part of such a memory system described above may be used tostore one or more data structures (e.g., look-up tables) or equationsdescribed above. For example, at least part of the non-volatilerecording medium may store at least part of a database that includes oneor more of such data structures. Such a database may be any of a varietyof types of databases, for example, a file system including one or moreflat-file data structures where data is organized into data unitsseparated by delimiters, a relational database where data is organizedinto data units stored in tables, an object-oriented database where datais organized into data units stored as objects, another type ofdatabase, or any combination thereof.

The signal processor/computer may include a video and audio data I/Osubsystem. An audio portion of the subsystem may include ananalog-to-digital (A/D) converter, which receives analog audioinformation and converts it to digital information. The digitalinformation may be compressed using known compression systems forstorage on the hard disk to use at another time. A typical video portionof the I/O subsystem may include a video image compressor/decompressorof which many are known in the art. Such compressor/decompressorsconvert analog video information into compressed digital information,and vice-versa. The compressed digital information may be stored on harddisk for use at a later time.

The signal processor/computer may include one or more output devices.Example output devices include a cathode ray tube (CRT), liquid crystaldisplays (LCD) and other video output devices, printers, communicationdevices such as a modern or network interface, storage devices such asdisk or tape, and audio output devices such as a speaker.

The signal processor/computer also may include one or more inputdevices. Example input devices include a keyboard, keypad, track ball,mouse, pen and tablet, communication devices such as described above,and data input devices such as audio and video capture devices andsensors. The signal processor/computer is not limited to the particularinput or output devices described herein.

The signal processor/computer may include specially programmed, specialpurpose hardware, for example, an application-specific integratedcircuit (ASIC). Such special-purpose hardware may be configured toimplement one or more of the methods, steps, simulations, algorithms,systems, and system elements described above. The signalprocessor/computer and components thereof may be programmable using anyof a variety of one or more suitable computer programming languages.Such languages may include procedural programming languages, forexample, C, Pascal, Fortran and BASIC, object-oriented languages, forexample, C++, Java and Eiffel and other languages, such as a scriptinglanguage or even assembly language. The methods, steps, simulations,algorithms, systems, and system elements may be implemented using any ofa variety of suitable programming languages, including proceduralprogramming languages, object-oriented programming languages, otherlanguages and combinations thereof, which may be executed by such acomputer system. Such methods, steps, simulations, algorithms, systems,and system elements can be implemented as separate modules of a computerprogram, or can be implemented individually as separate computerprograms. Such modules and programs can be executed on separatecomputers.

The methods, steps, simulations, algorithms, systems, and systemelements described above may be implemented in software, hardware orfirmware, or any combination of the three, as part of the computerimplemented control system described above or as an independentcomponent.

Such methods, steps, simulations, algorithms, systems, and systemelements, either individually or in combination, may be implemented as acomputer program product tangibly embodied as computer-readable signalson a computer-readable medium, for example, a non-volatile recordingmedium, an integrated circuit memory element, or a combination thereof.For each such method, step, simulation, algorithm, system, or systemelement, such a computer program product may comprise computer-readablesignals tangibly embodied on the computer-readable medium that defineinstructions, for example, as part of one or more programs, that, as aresult of being executed by a computer, instruct the computer to performthe method, step, simulation, algorithm, system, or system element.

Exemplary Target Analytes

As will be appreciated by those in the art, a large number of analytemolecules and particles may be detected and, optionally, quantifiedusing methods and systems of the present invention; basically, anyanalyte molecule that is able to be made to become immobilized withrespect to a binding ligand can be potentially investigated using theinvention. Certain more specific targets of potential interest that maycomprise an analyte molecule are mentioned below. The list below isexemplary and non-limiting.

In some embodiments, the analyte molecule may be a biomolecule.Non-limiting examples of biomolecules include hormones, antibodies,cytokines, proteins, nucleic acids, lipids, carbohydrates, lipidscellular membrane antigens and receptors (neural, hormonal, nutrient,and cell surface receptors) or their ligands, or combinations thereof.Non-limiting embodiments of proteins include peptides, polypeptides,protein fragments, protein complexes, fusion proteins, recombinantproteins, phosphoproteins, glycoproteins, lipoproteins, or the like. Aswill be appreciated by those in the art, there are a large number ofpossible proteinaceous analyte molecules that may be detected orevaluated for binding partners using the present invention. In additionto enzymes as discussed above, suitable protein analyte moleculesinclude, but are not limited to, immunoglobulins, hormones, growthfactors, cytokines (many of which serve as ligands for cellularreceptors), cancer markers, etc. Non-limiting examples of biomoleculesinclude PSA and TNF-alpha.

In certain embodiments, the analyte molecule may be ahost-translationally modified protein (e.g., phosphorylation,methylation, glycosylation) and the capture component may be an antibodyspecific to a post-translational modification. Modified proteins may becaptured with capture components comprising a multiplicity of specificantibodies and then the captured proteins may be further bound to abinding ligand comprising a secondary antibody with specificity to apost-translational modification. Alternatively, modified proteins may becaptured with capture components comprising an antibody specific for apost-translational modification and then the captured proteins may befurther bound to binding ligands comprising antibodies specific to eachmodified protein.

In another embodiment, the analyte molecule is a nucleic acid. A nucleicacid may be captured with a complementary nucleic acid fragment (e.g.,an oligonucleotide) and then optionally subsequently labeled with abinding ligand comprising a different complementary oligonucleotide.

Suitable analyte molecules and particles include, but are not limited tosmall molecules (including organic compounds and inorganic compounds),environmental pollutants (including pesticides, insecticides, toxins,etc.), therapeutic molecules (including therapeutic and abused drugs,antibiotics, etc.), biomolecules (including hormones, cytokines,proteins, nucleic acids, lipids, carbohydrates, cellular membraneantigens and receptors (neural, hormonal, nutrient, and cell surfacereceptors) or their ligands, etc), whole cells (including prokaryotic(such as pathogenic bacteria) and eukaryotic cells, including mammaliantumor cells), viruses (including retroviruses, herpesviruses,adenoviruses, lentiviruses, etc.), spores, etc.

In some embodiments, the analyte molecule may be an enzyme. Non-limitingexamples of enzymes include, an oxidoreductase, transferase, kinase,hydrolase, lyase, isomerase, ligase, and the like. Additional examplesof enzymes include, but are not limited to, polymerases, cathepsins,calpains, amino-transferases such as, for example, AST and ALT,proteases such as, for example, caspases, nucleotide cyclases,transferases, lipases, enzymes associated with heart attacks, and thelike. When a system/method of the present invention is used to detectthe presence of viral or bacterial agents, appropriate target enzymesinclude viral or bacterial polymerases and other such enzymes, includingviral or bacterial proteases, or the like.

In other embodiments, the analyte molecule may comprise an enzymaticcomponent. For example, the analyte particle can be a cell having anenzyme or enzymatic component present on its extracellular surface.Alternatively, the analyte particle is a cell having no enzymaticcomponent on its surface. Such a cell is typically identified using anindirect assaying method described below. Non-limiting example ofenzymatic components are horseradish peroxidase, beta-galactosidase, andalkaline phosphatase.

The fluid sample containing or suspected of containing an analytemolecule may be derived from any suitable source. In some cases, thesample may comprise a liquid, fluent particulate solid, fluid suspensionof solid particles, supercritical fluid, and/or gas. In some cases, theanalyte molecule may be separated or purified from its source prior todetermination; however, in certain embodiments, an untreated samplecontaining the analyte molecule may be tested directly. The source ofthe analyte molecule may be synthetic (e.g., produced in a laboratory),the environment (e.g., air, soil, etc.), a mammal, an animal, a plant,or any combination thereof. In a particular example, the source of ananalyte molecule is a human bodily substance (e.g., blood, serum,plasma, urine, saliva, tissue, organ, or the like). The volume of thefluid sample analyzed may potentially be any amount within a wide rangeof volumes, depending on a number of factors such as, for example, thenumber of capture objects used/available, the number of locationsus/available, etc. In a few particular exemplary embodiments, the samplevolume may be about 0.01 ul, about 0.1 uL, about 1 uL, about 5 uL, about10 uL, about 100 uL, about 1 mL, about 5 mL, about 10 mL, or the like.In some cases, the volume of the fluid sample is between about 0.01 uLand about 10 mL, between about 0.01 uL and about 1 mL, between about0.01 uL and about 100 uL, or between about 0.1 uL and about 10 uL.

In some cases, the fluid sample may be diluted prior to use in an assay.For example, in embodiments where the source of an analyte molecule is ahuman body fluid (e.g., blood, serum), the fluid may be diluted with anappropriate solvent (e.g., a buffer such as PBS buffer). A fluid samplemay be diluted about 1-fold, about 2-fold, about 3-fold, about 4-fold,about 5-fold, about 6-fold, about 10-fold, or greater, prior to use. Thesample may be added to a solution comprising the plurality of captureobjects, or the plurality of capture objects may be added directly to oras a solution to the sample.

Capture Components

In some embodiments of the present invention, the analyte molecules mayimmobilized with respect to a surface (e.g., the surface of a captureobject, the surface of a location (e.g., reaction vessel), or the like).The analyte molecules may be immobilized with respect to a surface priorto, concurrent with, or following exposure to a plurality of types ofbinding ligands. In some embodiments, immobilization of the analytemolecules with respect to a surface may aid in removal of any excessbinding ligands from the solution without concern of dislodging theanalyte molecule from the surface (e.g., from the reaction vessel).Generally, a capture component allows the attachment of a molecule,particle, or complex to a solid support (e.g., capture object, location,etc.) for the purposes of immobilization, detection, quantification,and/or other analysis of the molecule, particle, or complex.

As will be appreciated by those in the art, the composition of thecapture component will depend on the composition of the analytemolecule. Capture components for a wide variety of target molecules areknown or can be readily found or developed using known techniques. Forexample, when the target molecule is a protein, the capture componentsmay comprise proteins, particularly antibodies or fragments thereof(e.g., antigen-binding fragments (Fabs), Fab′ fragments, pepsinfragments, F(ab′)₂ fragments, full-length polyclonal or monoclonalantibodies, antibody-like fragments, etc.), other proteins, such asreceptor proteins, Protein A, Protein C, etc., or small molecules. Insome cases, capture components for proteins comprise peptides. Forexample, when the target molecule is an enzyme, suitable capturecomponents may include enzyme substrates and/or enzyme inhibitors. Insome cases, when the target analyte is a phosphorylated species, thecapture component may comprise a phosphate-binding agent. For example,the phosphate-binding agent may comprise metal-ion affinity media suchas those describe in U.S. Pat. No. 7,070,921 and U.S. Patent ApplicationNo. 20060121544. In addition, when the target molecule is asingle-stranded nucleic acid, the capture component may be acomplementary nucleic acid. Similarly, the target molecule may be anucleic acid binding protein and the capture component may be asingle-stranded or double-stranded nucleic acid; alternatively, thecapture component may be a nucleic acid-binding protein when the targetmolecule is a single or double stranded nucleic acid. Alternatively, asis generally described in U.S. Pat. Nos. 5,270,163, 5,475,096,5,567,588, 5,595,877, 5,637,459, 5,683,867, 5,705,337, and relatedpatents, nucleic acid “aptamers” may be developed for capturingvirtually any target molecule. Also, for example, when the targetmolecule is a carbohydrate, potentially suitable capture componentsinclude, for example, antibodies, lectins, and selectins. As will beappreciated by those of ordinary skill in the art, any molecule that canspecifically associate with a target molecule of interest maypotentially be used as a capture component.

For certain embodiments, suitable target analyte molecule/capturecomponent pairs can include, but are not limited to,antibodies/antigens, receptors/ligands, proteins/nucleic acid, nucleicacids/nucleic acids, enzymes/substrates and/or inhibitors, carbohydrates(including glycoproteins and glycolipids)/lectins and/or selectins,proteins/proteins, proteins/small molecules; small molecules/smallmolecules, etc. According to one embodiment, the capture components areportions (particularly the extracellular portions) of cell surfacereceptors that are known to multimerize, such as the growth hormonereceptor, glucose transporters (particularly GLUT 4 receptor), andT-cell receptors and the target analytes are one or more receptor targetligands.

In a particular embodiment, the capture component may be attached to thesurface via a linkage, which may comprise any moiety, functionalization,or modification of the binding surface and/or capture component thatfacilitates the attachment of the capture component to the surface. Thelinkage between the capture component and the surface may comprise oneor more chemical or physical (e.g., non-specific attachment via van derWaals forces, hydrogen bonding, electrostatic interactions,hydrophobic/hydrophilic interactions; etc.) bonds and/or chemicallinkers providing such bond(s). In certain embodiments, the capturecomponent comprises a capture extender component. In such embodiments,the capture component comprises a first portion that binds the analytemolecule and a second portion that can be used for attachment to thebinding surface.

In certain embodiments, a surface may also comprise a protective orpassivating layer that can reduce or minimize non-specific attachment ofnon-capture components (e.g., analyte molecules, binding ligands) to thebinding surface during the assay which may lead to false positivesignals during detection or to loss of signal. Examples of materialsthat may be utilized in certain embodiments to form passivating layersinclude, but are not limited to: polymers, such as poly(ethyleneglycol), that repel the non-specific binding of proteins; naturallyoccurring proteins with this property, such as serum albumin and casein;surfactants, e.g., zwitterionic surfactants, such as sulfobetaines;naturally occurring long-chain lipids; and nucleic acids, such as salmonsperm DNA.

One embodiment utilizes proteinaceous capture components. As is known inthe art, any number of techniques may be used to attach a proteinaceouscapture component to a wide variety of solid surfaces. “Protein” or“proteinaceous” in this context includes proteins, polypeptides,peptides, including, for example, enzymes, and antibodies. A widevariety of techniques are known to add reactive moieties to proteins,for example, the method outlined in U.S. Pat. No. 5,620,850. Theattachment of proteins to surfaces is known, for example, see Heller,Acc. Chem. Res. 23:128 (1990), and many other similar references.

In some embodiments, the capture component (or binding ligand) maycomprise Fab′ fragments. The use of Fab′ fragments as opposed to wholeantibodies may help reduce non-specific binding between the capturecomponent and the binding ligand. In some cases, the Fc region of acapture component (or binding ligand) may be removed (e.g.,proteolytically). In some cases, an enzyme may be used to remove the Fcregion (e.g., pepsin, which may produce F(ab′)₂ fragments and papain,which may produce Fab fragments). In some instances, the capturecomponent may be attached to a binding surface using amines or may bemodified with biotin (e.g., using NHS-biotin) to facilitate binding toan avidin or streptavidin coated capture object surface. F(ab′)₂fragments may be subjected to a chemical reduction treatment (e.g., byexposure to 2-mercaptoethylamine) to, in some cases, form twothiol-bearing Fab′ fragments. These thiol-bearing fragments can then beattached via reaction with a Michael acceptor such as maleimide. Forexample, the Fab′ fragments may then be treated with a reagent (e.g.,maleimide-biotin) to attach at least one biotin moiety (i.e.,biotinylated) to facilitate attachment to streptavidin-coated surfacesas described above.

Certain embodiments utilize nucleic acids as the capture component, forexample for when the analyte molecule is a nucleic acid or a nucleicacid binding protein, or when the it is desired that the capturecomponent serve as an aptamer for binding a protein, as is well known inthe art.

According to one embodiment, each binding surface comprises a pluralityof capture components. The plurality of capture components, in somecases, may be distributed randomly on the binding surface like a “lawn.”Alternatively, the capture components may be spatially segregated intodistinct region(s) and distributed in any desired fashion.

Binding between the capture component and the analyte molecule, incertain embodiments, is specific, e.g., as when the capture componentand the analyte molecule are complementary parts of a binding pair. Incertain such embodiments, the capture component binds both specificallyand directly to the analyte molecule. By “specifically bind” or “bindingspecificity,” it is meant that the capture component binds the analytemolecule with specificity sufficient to differentiate between theanalyte molecule and other components or contaminants of the testsample. For example, the capture component, according to one embodiment,may be an antibody that binds specifically to some portion of an analytemolecule (e.g., an antigen). The antibody, according to one embodiment,can be any antibody capable of binding specifically to an analytemolecule of interest. For example, appropriate antibodies include, butare not limited to, monoclonal antibodies, bispecific antibodies,minibodies, domain antibodies, synthetic antibodies (sometimes referredto as antibody mimetics), chimeric antibodies, humanized antibodies,antibody fusions (sometimes referred to as “antibody conjugates”), andfragments of each, respectively. As another example, the analytemolecule may be an antibody and the capture component may be an antigen.

According to one embodiment in which an analyte particle is a biologicalcell (e.g., mammalian, avian, reptilian, other vertebrate, insect,yeast, bacterial, cell, etc.), the capture component may be a ligandhaving specific affinity for a cell surface antigen (e.g., a cellsurface receptor). In one embodiment, the capture component is anadhesion molecule receptor or portion thereof, which has bindingspecificity for a cell adhesion molecule expressed on the surface of atarget cell type. In use, the adhesion molecule receptor binds with anadhesion molecule on the extracellular surface of the target cell,thereby immobilizing or capturing the cell. In one embodiment in whichthe analyte particle is a cell, the capture component is fibronectin,which has specificity for, for example, analyte particles comprisingneural cells.

In some embodiments, as will be appreciated by those of ordinary skillin the art, it is possible to detect analyte molecules using capturecomponents for which binding to analyte molecules that is not highlyspecific. For example, such systems/methods may use different capturecomponents such as, for example, a panel of different binding ligands,and detection of any particular analyte molecule is determined via a“signature” of binding to this panel of binding ligands, similar to themanner in which “electronic noses” work. This may find particularutility in the detection of certain small molecule analytes. In someembodiments, the binding affinity between analyte molecules and capturecomponents should be sufficient to remain bound under the conditions ofthe assay, including wash steps to remove molecules or particles thatare non-specifically bound. In some cases, for example in the detectionof certain biomolecules, the binding constant of the analyte molecule toits complementary capture component may be between at least about 10⁴and about 10⁶ M⁻¹, at least about 10⁵ and about 10⁹ M⁻¹, at least about10⁷ and about 10⁹ M⁻¹, greater than about 10⁹ M⁻¹, or greater.

Binding Ligands and Precursor Labeling Agents/Labeling Agent

In some embodiment, the assay may comprise the use of at least onebinding ligand. Binding ligands may be selected from any suitablemolecule, particle, or the like, as discussed more below, able toassociate with an analyte molecule and/or to associate with anotherbinding ligand. Certain binding ligands can comprise a component that isable to facilitate detection, either directly (e.g., via a detectablemoiety) or indirectly. A component may facilitate indirect detection,for example, by converting a precursor labeling agent molecule into alabeling agent molecule (e.g., an agent that is detected in an assay).In some embodiments, the binding ligand may comprise an enzymaticcomponent (e.g., horseradish peroxidase, beta-galactosidase, alkalinephosphatase, etc). A first type of binding ligand may or may not be usedin conjunction with additional binding ligands (e.g., second type,etc.), as discussed herein.

In some embodiments, the plurality of analyte molecules (e.g., in somecases, immobilized with respect to a capture object) may be exposed to aplurality of binding ligands such that a binding ligand associates withat least some of the plurality of analyte molecules. In some cases,greater than about 80%, greater than about 85%, greater than about 90%,greater than about 95%, greater than about 97%, greater than about 98%,greater than about 99%, or more, analyte molecules associate with abinding ligand.

For the capture step, the choice of bead concentration may depend onseveral competing factors. For example, it can be advantageous ifsufficient beads are present to capture most of the target analyte fromthermodynamic and kinetic perspectives. As an exemplary illustration,thermodynamically, 200,000 beads in 100 μL that each have about 80,000capture components (e.g. antibodies) bound to correlates to an antibodyconcentration of about 0.3 nM, and the antibody-protein equilibrium atthat concentration may give rise to a relatively high capture efficiencyof target analyte molecules in certain cases (e.g. >70%). Kinetically,for 200,000 beads dispersed in 100 μL, the average distance betweenbeads can be estimated to be about 80 μm. Proteins the size of TNF-α andPSA (17.3 and 30 kDa, respectively), as exemplary analyte molecules, forexample, will typically tend to diffuse 80 μm in less than 1 min, suchthat, over a 2 hour incubation, capture of such analyte molecules willtend not to be limited kinetically. In addition, it can also beadvantageous to provide sufficient beads loaded onto the arrays to limitPoisson noise to a desired or acceptable amount. Considering as anexample a situation where 200,000 beads in a in 10 μL volume are loadedonto an array, typically about 20,000-30,000 beads may become trapped infemtoliter sized wells of the array. For a typical background signal(e.g. due to non specific binding, etc.) of 1% active beads, thisloading would be expected to result in a background signal of 200-300active beads detected, corresponding to a coefficient of variation (CV)from Poisson noise of 6-7%, which may be acceptable in typicalembodiments. However, bead concentrations above certain concentrationsmay be undesirable in certain cases in that they may lead to: a)increases in non-specific binding that may reduce signal-to-background;and/or b) undesirably low ratios of analyte-to-bead such that thefraction of active beads is too low, resulting in high CVs from Poissonnoise. In certain embodiments, considering a balance of factors such asthose discussed above, providing about 200,000 to 1,000,000 beads per100 μL of test sample may be desirable or, in certain cases optimal, forperforming certain assays of the invention.

For embodiments of the inventive assay employing one or more bindingligand(s) to label the captured analyte molecules, it may beadvantageous to, in certain instances, adjust the concentrations used toyield desirable or optimal performance. For example, considering anembodiment involving an analyte molecule that is a protein (capturedprotein) and employing a first binding ligand comprising a detectionantibody and a second binding ligand comprising an enzyme conjugate(e.g. SβG), the concentrations of detection antibody and enzymeconjugate (SβG) used to label the captured protein may in some cases belimited or minimized to yield an acceptable background signal (e.g. 1%or less) and Poisson noise. The choice of the concentrations ofdetection antibody and enzyme conjugate (SβG) used to label the capturedprotein can be factors in improving the performance of or optimizingcertain of the inventive assay methods. In certain cases, it may bedesirable for only a fraction of the capture proteins to be labeled soas to avoid saturating signals produced by the assay. For example, for aparticular assay where background levels observed are equivalent to ˜1-2fM of target protein, such that the ratio of analyte to bead may beabout 0.3-0.6, the number of active beads may be in the range of about25-40% if every protein was labeled with an enzyme, which may be higherthan desirable in some cases. To produce background signals that may becloser to a lower end of the dynamic range for a digital detectionassay—considering e.g. that in certain cases 1% active beads may providea reasonable noise floor for background in digital detection assays ofthe invention—appropriate labeling of the captured protein canpotentially be achieved by kinetic control of the labeling steps, eitherby limiting or minimizing the concentrations of both labeling reagentsor by using shorter incubation times. For example, in an embodimentwhere label concentrations are minimized, use of a standard ELISAincubation time may provide acceptable results; e.g. using a total assaytime of ˜6 h. This length of time may be acceptable for testing thattolerates a daily turnaround time for samples. For shorter turnaroundtimes of, for example, <1 hour (e.g., for point-of-care applications),the assay could be performed with shorter incubations with higherconcentrations of labels.

In some embodiments, more than one type of binding ligand may be used.In some embodiments, a first type of binding ligand and a second type ofbinding ligand may be provided. In some instances, at least two, atleast three, at least four, at least five, at least eight, at least ten,or more, types of binding ligands may be provided. When a plurality ofcapture objects, some of which are associated with at least one analytemolecule, are exposed to a plurality of types of binding ligand, atleast some of the plurality of immobilized analyte molecules mayassociate with at least one of each type of binding ligand. The bindingligands may be selected such that they interact with each other in avariety of different manners. In a first example, the first type ofbinding ligand may be able to associate with an analyte molecule and thesecond type of binding ligand may be able to associate with the firsttype of binding ligand. In such embodiments, the first type of bindingligand may comprise a first component which aids in association of theanalyte molecule and a second component which aids in association of thesecond type of binding ligand with the first type of binding ligand. Ina particular embodiment, the second component is biotin and the secondtype of binding ligand comprises an enzyme or an enzymatic componentwhich associates with the biotin.

As another example, both the first type of binding ligand and the secondtype of binding ligand may associate directly with an analyte molecule.Without being bound by theory or any particular mechanism, theassociation of both the first type and the second type of binding ligandmay provide additional specificity and reliability in performing anassay, by identifying only locations which are determined to containboth the first type of binding ligand and/or the second type of bindingligand (e.g., either through direct or indirect detection) as containingan analyte molecule. Such assay methods may reduce the number of falsepositives caused by non-specific binding as locations that are found toonly have a single type of binding ligand (e.g., only the first type oflabeling agent or the second type of labeling agent) would be not beconsidered or counted as a location comprising an analyte molecule. Thefirst type of binding ligand may comprise a first type of enzymaticcomponent and the second type of binding ligand may comprise a secondtype of enzymatic component which differs from the first type ofenzymatic component. A capture object comprising an analyte molecule,the first type of binding ligand, and the second type of binding ligandmay be exposed to a first type of precursor labeling agent which isconverted to a first type of labeling agent (e.g., comprising a firstmeasurable property) upon exposure to the first type of enzymaticcomponent and a second type of precursor labeling agent which isconverted to a second type of labeling agent (e.g., comprising ameasurable property which is distinguishable from the first measurableproperty) upon exposure to the second type of enzymatic component.Therefore, only locations which are determined to contain the first typeof labeling agent and the second type of labeling agent are determinedto contain an analyte molecule. As another example, the first type ofbinding ligand and the second type of binding ligand may eachincorporate a component (e.g., such as a DNA label) and a third type ofbinding ligand may comprise two components complimentary to thecomponents of the first type and second type of binding ligands (e.g.,two types of complimentary DNA labels), wherein the third type ofbinding ligand also comprises an molecule or moiety for direct orindirect detection (e.g., the presence of the third type of bindingligand in a reaction vessel is required to determine the presence orabsence of an analyte molecule in a location). When both the first typeof binding ligands and the second types of binding ligands are presentin substantially close proximity to each other (e.g., via associationwith an analyte molecule) association of the third type of bindingligand may occur, and therefore, for detection of the analyte molecule.More information regarding the use of more than one type of bindingligand in a manner which may reduce certain negative affects associatedwith non-specific binding, are described in commonly owned U.S. patentapplication Ser. No. 12/731,135, entitled “Ultra-Sensitive Detection ofMolecules using Dual Detection Methods” by Duffy et al., filed Mar. 24,2010; and International Patent Application Serial No. PCT/US11/026,657,entitled “Ultra-Sensitive Detection of Molecules using Dual DetectionMethods” by Duffy et al., filed Mar. 1, 2011, incorporated by reference.

The following examples are included to demonstrate various features ofthe invention. Those of ordinary skill in the art should, in light ofthe present disclosure, will appreciate that many changes can be made inthe specific embodiments which are disclosed while still obtaining alike or similar result without departing from the scope of the inventionas defined by the appended claims. Accordingly, the following examplesare intended only to illustrate certain features of the presentinvention, but do not necessarily exemplify the full scope of theinvention.

Example 1

The following example describes materials used in Examples 2-10. Opticalfiber bundles were purchased from Schott North America (Southbridge,Mass.). Non-reinforced gloss silicone sheeting was obtained fromSpecialty Manufacturing (Saginaw, Mich.). Hydrochloric acid, anhydrousethanol, and molecular biology grade Tween-20 were purchased fromSigma-Aldrich (Saint Louis, Mo.). 2.7-μm-diameter carboxyl-terminatedmagnetic beads were purchased from Varian, Inc. (Lake Forest, Calif.).1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC),N-hydroxysulfosuccinimide (NHS), and SuperBlock® T-20 Blocking Bufferwere purchased from Thermo Scientific (Rockford, Ill.).Streptavidin-β-galactosidase (SβG) was purchased from Invitrogen,Sigma-Aldrich, or conjugated in house using standard protocols.Resorufin-3-D-galactopyranoside (RGP) was purchased from Invitrogen(Carlsbad, Calif.). The fiber polisher and polishing consumables werepurchased from Allied High Tech Products (Rancho Dominguez, Calif.).Monoclonal capture antibody to PSA, monoclonal detection antibody toPSA, and purified PSA were purchased from BiosPacific. The Chromalink™biotinylation reagent was purchased from Solulink, Inc (San Diego,Calif.). Purified DNA was purchased from Integrated DNA Technologies.

Example 2

The following describes a non-limiting example of the preparation of2.7-um-diameter magnetic beads functionalized with biotin for capture ofthe exemplary analyte, SβG. Beads functionalized with DNA capture probe(5′-NH₂/C12-GTT GTC AAG ATG CTA CCG TTC AGA G-3′) (SEQ ID NO: 1) wereprepared according to the manufacturer's instructions. These beads wereincubated with 1 μM of biotinylated complementary DNA (5′-biotin-C TCTGAA CGG TAG CAT CTT GAC AAC-3′) (SEQ ID NO: 2) overnight (16 h) in TEbuffer containing 0.5M NaCl and 0.01% Tween-20. After incubation, thebeads were washed three times in PBS buffer containing 0.1% Tween-20.The bead stock was distributed into a microtiter plate giving 400,000beads per well in 100 μL. The buffer was aspirated from the microtiterplate wells, the beads were resuspended and incubated with variousconcentrations of SβG in Superblock containing 0.05% Tween-20 for 5 h.The beads were then separated and washed six times with 5× PBS buffercontaining 0.1% Tween-20.

In an alternative embodiment, a bead concentration of 200,000 beads per100 μL of SβG target solution was used. Beads were resuspended andincubated with various concentrations of SβGin target solutions dilutedin SuperblockSuperBlock containing 0.05% Tween-20 for 54 h. 100 μL ofthe various target solutions were aliquotted into a microtiter plate.The beads were then separated with a microtiter plate magnet and washedsix times with 5×PBS buffer containing 0.1% Tween-20. For detection, thebeads were resuspended in 10 μL of PBS containing 0.1% Tween-20, and thealiquots were loaded onto a femtoliter-volume well array.

Example 3

The following describes a non-limiting example of the preparation ofmicrowells arrays. Optical fiber bundles approximately 5-cm long weresequentially polished on a polishing machine using 30-, 9-, and1-micron-sized diamond lapping films. The polished fiber bundles werechemically etched in a 0.025 M HCl solution for 130 seconds, and thenimmediately submerged into water to quench the reaction. On average, thewells etch at a rate of approximately 1.5 to 1.7 μm per minute.Therefore, wells of 3.25 um depth are produced in about 115 to 130 s. Toremove impurities from etching, the etched fibers were sonicated for 5 sand washed in water for 5 min. The fibers were then dried under vacuumand exposed to air plasma for 5 min to clean and activate the glasssurface. The arrays were silanized for 30 minutes in a 2% solution ofsilane to make the surfaces hydrophobic. In some cases, the dimensionsof the wells (e.g., 3.25±0.5 m) were configured for retaining singlebeads in wells while maintaining good seals.

Example 4

The following describes a non-limiting example of the loading of beadsinto microwells. Prior to loading of the beads into the etched wells,the beads may be exposed to the fluid sample comprising the analytemolecules. To apply the solution of beads to the etched wells in a fiberbundle, clear PVC tubing ( 1/16″ I.D. ⅛″ O.D.) and clear heat shrink (3/16″ ID) were cut into approximately 1 cm long. A piece of PVC tubingwas first place on the etched end of a fiber bundle to create areservoir to hold the bead solution, followed by the application of heatshrink around the interface between the PVC tubing and fiber bundle toprovide a tight seal. 10 uL of the concentrated bead solution waspipetted into the reservoir created by the PVC tubing. The fiber bundlewas then centrifuged at 3000 rpm (˜1300 g) for 10 minutes to force thebeads into the etched wells. The PVC tubing/heat shrink assembly wasremoved after centrifugation. The distal end of the fiber bundle wasdipped in PBS solution to wash off excess bead solution, followed byswabbing the surface with deionized water. In embodiments where the beadconcentrations was 200,000 per 10 μL, this typically resulted in 40-60%of wells in a 50,000-well array being occupied by a single bead.

Example 5

The following describes a non-limiting example of the loading anddetection of beads and enzyme-labeled beads in microwell arrays. Acustom-built imaging system containing a mercury light source, filtercubes, objectives, and a CCD camera was used for acquiring fluorescenceimages. Fiber bundle arrays were mounted on the microscope stage using acustom fixture. A droplet of β-galactosidase substrate (RPG) was placedon the silicone gasket material, and put into contact with the distalend of the fiber array. The precision mechanical platform moved thesilicone sheet into contact with the distal end of the etched opticalfiber array, creating an array of isolated femtoliter reaction vessels.Fluorescence images were acquired at 577 nm with an exposure time 1011ms. Five frames (at 30 seconds per frame) were taken for each fiberbundle array. The fluorescent images were analyzed using image analysissoftware to determine the presence or absence of enzymatic activitywithin each well of the microwell array. The data was analyzed using adeveloped image processing software using MathWorks MATLAB and MathWorksImage Processing toolbox. The software aligns acquired image frames,identifies reaction vessel positions, locates reaction vessels withbeads and measures the change in reaction vessel intensity over apredefined time period. Reaction vessels containing beads withsufficient intensity growth over all data frames are counted and thefinal number of active reaction vessels is reported as a percentage ofall identified reaction vessels

As well as fluorescence, the arrays were imaged with white light toidentify those wells that contain beads. After acquiring thefluorescence images, the distal (sealed) end of the fiber bundle arrayswere illuminated with white light and imaged on the CCD camera. Due toscattering of light by the beads, those wells that contained a beadappeared brighter in the image than wells without beads. Beaded wellswere identified using this method by software.

Example 6

The following non-limiting method describes extending the dynamic rangeof single molecule measurements. The experiment described above inExample 5 was repeated across a wide range of enzyme concentrations(e.g., see Table 2 for range of concentrations). The images generated inthis experiment were analyzed in different ways depending on theconcentration tested. For example, as described in the above in thedetailed description, when the percentage of beads associated with atleast one analyte molecules (e.g., enzymes) was less than about 50% (or45%, or 40%, or 35%, etc.) the average molecule per bead was determinedby counting the total number of “on” beads. An “on” bead was identifiedas a well that contained a bead (from the white light image), and whosefluorescence increased in all four consecutive frames after the firstframe, and whose overall fluorescence increased by at least 20% from thefirst frame to the last. The total number of “on” beads may be adjustedusing a Poisson distribution adjustment. At high ratios of “on” beads,the average bead signal was determined from the intensity of the secondframe captured. The analog-to-digital conversion factor was determinedusing a sample of known concentration which had an “on” bead percentagebetween about 30% and about 50%. The results for the samples of knownconcentration may be plotted on a calibration curve, with aid of acalibration factor. Using the resulting calibration curve, the unknownconcentration of analyte molecules in a fluid sample may be determined.For example, by addressing at least some of the plurality of locationscontaining at least one bead and determining a measure indicative of thepercentage of said locations containing at least one analyte molecule orparticle (e.g., the percentage of “on” beads). Depending on thepercentage, a measure of the unknown concentration of analyte moleculesor particles in the fluid sample may be determined based at least inpart on the percentage or based at least in part on a measured intensityof a signal that is indicative of the presence of a plurality of analytemolecules or particles, and by comparison of the value with thecalibration curve.

Example 7

The following example describes the preparation and characterization ofbiotinylated PSA detection antibodies and enzyme conjugates. Detectionantibodies were biotinylated using the Chromalink™ biotinylationreagent. This reagent contains a succinimidyl ester group that attachesbiotin groups to the antibody via lysine residues, and a bis arylhydrazone chromophore that allows quantification of the number of biotinmolecules per antibody. The average number of biotin groups on theanti-PSA antibody ranged from 7.5 to 9.5. Streptavidin-β-galactosidase(SβG) was conjugated using standard protocols. HPLC characterization ofthe conjugate indicated that >80% of the conjugate molecules containedone β-galactosidase molecule, with an average of 1.2 enzymes/conjugate.Comparison to molecular weight standards indicated that the averagenumber of streptavidin molecules conjugated to each enzyme molecule was2.7. As each detection antibody contains multiple biotin groups, it ispossible that a single protein molecule bound to a single detectionantibody could be bound to multiple enzyme conjugates. Analysis of thefluorescence intensity generated by enzyme-associated beads in thesingle molecule regime (AMB<0.1) suggests, however, that multiple enzymeconjugates did not bind to single detection antibody molecules: thesefluorescent intensities were consistent with the previously-knownkinetics of single molecules of β-galactosidase.

Example 8

The following example describes the capture of PSA on magnetic beads andformation of enzyme-labeled immunocomplexes. Beads functionalized with amonoclonal antibody to PSA were prepared according to the manufacturer'sinstructions. Test solutions (100 μL) were incubated with suspensions of500,000 magnetic beads for 2 h at 23° C. The beads were then separatedand washed three times in 5×PBS and 0.1% Tween-20. The beads wereresuspended and incubated with solutions containing detection antibody(˜1 nM) for 60 min at 23° C. The beads were then separated and washedthree times in 5×PBS and 0.1% Tween-20. The beads were incubated withsolutions containing SβG (15 pM) for 30 min at 23° C., separated andwashed seven times in 5×PBS and 0.1% Tween-20. The beads were thenresuspended in 25 μL of PBS, and 10 μL of the bead solution was loadedonto a femtoliter-volume well array.

Example 9

The following example describes an exemplary system where a combineddigital and analog enzyme label detection was carried out using theanalysis described above in the context of the second analog analysisembodiment (Equation 8). The SβG binding assay described above inExample 7 and using the methods and apparatus described in the otherexamples demonstrated an extended dynamic range can be achieved bycombining digital and analog determination of AMB. FIG. 13 shows AMBdetermined from images of populations of biotin-presenting beads thathad been incubated with concentrations of SβG ranging from zeptomolar topicomolar. Specifically, FIGS. 13A and 13B show that a broad dynamicrange was achieved by combining digital and analog measurements. FIG.13A shows a plot of AMB as a function of enzyme concentration. The errorbars are standard deviations over three replicates. FIG. 13B shows atable including the % active and AMB values as a function of enzymeconcentration. AMB was determined using Equation 4 for % active <70% andAMB was determined using Equation 8 for % active >70%. The thresholdbetween analog and digital in this example was between 10 fM and 31.6fM.

For images with % active beads <70%, AMB_(digital) was determined usingEquation 4. All the arrays with <10% active beads were used to determineĪ_(single), a total of 7566 beads; Ī_(single) was equal to 298 au. Theaverage fluorescence intensities of beads in images with over 70% activewere determined, and AMB_(analog) values were calculated using Equation8. Because the 0 M SβG concentration yielded no active beads, the lowerlimit of detection in this experiment could not be calculated using thebackground plus 3 s.d. method. Using a previously established LOD of 220zM, and the highest concentration detected in the linear range of thiscurve, 316 fM, a 6.2-log linear dynamic range for detecting enzyme labelwas observed. The linear digital dynamic range was 4.7 logs and theanalog linear dynamic range was 1.5 logs.

Example 10

This example describes a combined digital and analog system and methodfor measuring PSA in serum. The combined digital and analog approach wasused for determining concentration of PSA with a wide dynamic range.Clinically, PSA is used to screen for prostate cancer and to monitor forbiochemical recurrence of the disease in patients who have undergonesurgery to remove the cancer. The PSA levels in the serum of patientswho have undergone radical prostatectomy (RP) are known to range from0.014 pg/mL to over 100 pg/mL. To successfully measure the PSA levels inthe majority of patients in a single test requires an assay with 4 logsof dynamic range. The experiments were conducted as described in Example8. FIGS. 14A and 14B show that by combining digital and analog analyses,the working range of the inventive PSA assay ranged from 0.008 pg/mL to100 pg/mL, enabling precise quantification of PSA levels in the vastmajority of RP patient samples in one pass. Specifically, FIGS. 14A and14B show combined digital and analog PSA assay results with a 4-logworking range and calculated LOD of 0.008 pg/mL. AMB is plotted as afunction of PSA concentration in (FIG. 14A) linear-linear space and(FIG. 14B) log-log space. Error bars are shown for all data points basedon quadruplicate measurements.

The assay was used to measure the concentration of PSA in the sera of 17prostate cancer patients collected at 2 to 46 weeks (mean=13.8 weeks)after radical prostatectomy surgery. These samples were collected closeto surgery in order to push the lower limits of detection of digitalELISA. Here, in order to evaluate the dynamic range of the assay acrossthe intended clinical range, samples collected closer to surgery weretested to capture patients with higher PSA whose cancer could recur. PSAwas, however, undetectable in all of these samples using a leading PSAdiagnostic test (Siemens). Serum samples were diluted 1:4 in buffer andthe AMB was measured using the methods described herein. Theconcentration of PSA for each sample was determined by reading the AMBoff a simultaneously acquired calibration curve similar to FIG. 14B.Table 4 summarizes the AMB and PSA concentrations determined from thesesamples, along with the imprecision for signal and concentration givenby % CV. PSA was quantified in all of the samples in one experiment. Theaverage PSA concentration in these samples was 33 pg/mL, with a high of136 pg/mL and a low of 0.4 pg/mL. When combined with previousmeasurements of PSA in patient who had undergone radical prostatectomy(RP) surgery, the detected concentrations of PSA in clinical samplesranged from 0.46 fM (0.014 pg/mL) to 4.5 pM (136 pg/mL), demonstratingthe importance of the good dynamic range provided by the inventive assayof this example.

TABLE 4 Summary of AMB and [PSA] determined for 17 serum samples frompost-RP patients^(a) sample mean std dev AEB CV, [PSA] std dev [PSA] IDAEB AEB % (pg/mL) [PSA] CV, % S640 8.8 1.2 13 41.0 7.5 18 S641 0.87 0.0810 4.1 0.4 11 S643 0.23 0.002 1 1.1 0.01 1 S644 15.2 1.0 6 136 13 10S645 1.5 0.06 4 6.1 0.2 4 S647 11.0 0.1 1 80.4 1.9 2 S648 7.4 0.3 3 32.31.6 5 S649 1.5 0.2 11 6.1 0.6 10 S650 0.22 0.008 4 1.2 0.03 2 S651 0.500.02 5 2.3 0.1 4 S615 12.6 1.0 8 70.7 8.6 12 S653 1.3 0.2 18 5.0 0.8 16S616 0.44 0.05 12 1.7 0.2 13 S618 13.6 0.7 5 79.2 6.2 8 S624 14.8 0.5 388.9 4.1 5 S627 0.098 0.003 3 0.39 0.01 3 S628 0.92 0.04 5 3.7 0.15 4^(a)Standard deviations and CVs were determine over triplicate tests.Sample S644 had an AEB value beyond the range of the calibration curve,but its concentration was determined by extrapolation.

Example 11

FIG. 15 demonstrates a non-limiting example of a Poisson distributionanalysis in the digital range of a calibration curve using an assay forstreptavidin-β-galactosidase (SβG) that resulted in beads havingwell-defined enzyme/bead ratios. Briefly, beads were functionalized witha biotinylated capture molecule, and these beads were used to capturevarious concentrations of the SβG enzyme conjugate. The beads wereloaded into the femtoliter arrays and, after sealing a solution of RGPinto the wells of the array, fluorescence was generated from boundenzymes accumulated in the reaction chambers for 2.5 min, withfluorescent images acquired every 30 s. A white light image of the arraywas acquired at the end of the experiment. These images were analyzed toidentify wells that contained beads (from the white light image) anddetermine which of those beads had associated bound enzyme molecules(from time-lapsed fluorescent images. FIG. 15B shows that AMB_(digital)determined from Equation 4 maintained a linear response up to 50%active, despite non-linear variation in f_(on). Specifically, FIG. 15shows (FIG. 15A) conversion of % active beads to AMB_(digital) usingPoisson statistics. The center column is the fraction of active beadsdetermined by digital counting as a function of enzyme concentration.The right column is the average enzyme per bead (AMB) determined from %active beads using Equation 4. This conversion accounts statisticallyfor beads associated with multiple enzyme molecules using a digitalcounting method; (FIG. 15B) shows a plot of % active beads (diamonds)and AMB_(digital) (squares) as a function of enzyme concentration. The %active beads deviates from linearity with increasing concentration asexpected from the Poisson distribution. In this experiment,AMB_(digital) was linear with concentration up to about 50% activebeads. Error bars were determined from the standard deviation over threemeasurements.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively.

What is claimed:
 1. A system for determining a measure of theconcentration of analyte molecules or particles in a fluid sample,comprising: at least one detector configured to address locations of anassay substrate, able to produce at least one signal indicative of thepresence or absence of an analyte molecule or particle at each locationaddressed and having an intensity varying with the number of analytemolecules or particles at each location; and at least one signalprocessor configured to determine from the at least one signal a measureindicative of the number or percentage of said locations containing atleast one analyte molecule or particle, and further configured to, basedupon the measure indicative of the number or percentage, eitherdetermine a measure of the concentration of analyte molecules orparticles in the fluid sample based at least in part on the measureindicative of the number or percentage of locations containing at leastone analyte molecule or particle, or determine a measure of theconcentration of analyte molecules or particles in the fluid samplebased at least in part on an intensity level of the at least one signalindicative of the presence of a plurality of analyte molecules orparticles.
 2. The system of claim 1, wherein when the percentage of saidlocations containing at least one analyte molecule or particle is lessthan about 40%, less than about 35%, or less than about 30%, the measureof the concentration of analyte molecules or particles in the fluidsample is based at least in part on the measure indicative of the numberor percentage of locations containing at least one analyte molecule orparticle.
 3. The system of claim 1, wherein when the percentage of saidlocations containing at least one analyte molecule or particle is lessthan about 80%, less than about 75%, less than about 70%, less thanabout 65%, or less than about 60%, the measure of the concentration ofanalyte molecules or particles in the fluid sample is based at least inpart on the measure indicative of the number or percentage of locationscontaining at least one analyte molecule or particle.
 4. The system ofclaim 1, wherein when the percentage of said locations containing atleast one analyte molecule or particle is greater than about 30%,greater than about 35%, greater than about 40%, or greater than about45%, the measure of the concentration of analyte molecules or particlesin the fluid sample is based at least in part on an intensity level ofthe at least one signal indicative of the presence of a plurality ofanalyte molecules or particles.
 5. The system of claim 1, wherein whenthe percentage of said locations containing at least one analytemolecule or particle is greater than about 60%, greater than about 65%,greater than about 70%, or greater than about 75%, the measure of theconcentration of analyte molecules or particles in the fluid sample isbased at least in part on an intensity level of the at least one signalindicative of the presence of a plurality of analyte molecules orparticles.
 6. The system of claim 1, wherein when the percentage of saidlocations containing at least one analyte molecule or particle isbetween about 30% and about 50%, or between about 35% and about 45%, orabout 40%, the measure of the concentration of analyte molecules orparticles in the fluid sample is an average of the measure of theconcentration of analyte molecules or particles in the fluid samplebased at least in part on the measure indicative of the number orpercentage of locations containing at least one analyte molecule orparticle and the measure of the concentration of analyte molecules orparticles in the fluid sample based at least in part on an intensitylevel of the at least one signal indicative of the presence of aplurality of analyte molecules or particles.
 7. The system of claim 1,wherein when the percentage of said locations containing at least oneanalyte molecule or particle is between about 60% and about 80%, orbetween about 65% and about 75%, or about 70%, the measure of theconcentration of analyte molecules or particles in the fluid sample isan average of the measure of the concentration of analyte molecules orparticles in the fluid sample based at least in part on the measureindicative of the number or percentage of locations containing at leastone analyte molecule or particle and the measure of the concentration ofanalyte molecules or particles in the fluid sample based at least inpart on an intensity level of the at least one signal indicative of thepresence of a plurality of analyte molecules or particles.
 8. The systemof claim 1, further comprising the assay substrate operatively coupledto the at least one detector.
 9. The system of claim 8, wherein thelocations of the assay substrate each comprise a binding surface formingor contained within such locations.
 10. The system of claim 9, whereinat least one binding surface comprises at least one analyte molecule orparticle immobilized on the binding surface.
 11. The system of claim 9,wherein the binding surface is contained within such locations.
 12. Thesystem of claim 11, wherein the binding surface comprises a captureobject.
 13. The system of claim 12, wherein the capture object comprisesa bead.
 14. The system of claim 12, wherein the locations addressed arelocations which contain at least one capture object.
 15. The system ofclaim 1, wherein the at least one signal processor is configured todetermine from the at least one signal a measure indicative of thenumber of said locations containing at least one analyte molecule orparticle, and is further configured to, based upon the measureindicative of the number, either determine a measure of theconcentration of analyte molecules or particles in the fluid samplebased at least in part on the measure indicative of the number oflocations containing at least one analyte molecule or particle, ordetermine a measure of the concentration of analyte molecules orparticles in the fluid sample based at least in part on an intensitylevel of the at least one signal indicative of the presence of aplurality of analyte molecules or particles.
 16. The system of claim 1,wherein the at least one signal processor is configured to determinefrom the at least one signal a measure indicative of the percentage ofsaid locations containing at least one analyte molecule or particle, andis further configured to, based upon the measure indicative of thepercentage, either determine a measure of the concentration of analytemolecules or particles in the fluid sample based at least in part on themeasure indicative of the percentage of locations containing at leastone analyte molecule or particle, or determine a measure of theconcentration of analyte molecules or particles in the fluid samplebased at least in part on an intensity level of the at least one signalindicative of the presence of a plurality of analyte molecules orparticles.
 17. The method of claim 1, wherein the at least one signalprocessor is configured such that when the measure of the concentrationof analyte molecules or particles in the fluid sample is based at leastin part on the measure indicative of the number of locations containingat least one analyte molecule or particle, the measure of theconcentration is based at least in part on a ratio of the number of saidlocations associated with at least one analyte molecule or particle ofthe fluid sample to the number of locations not associated with ananalyte molecule or particle of the fluid sample.
 18. The method ofclaim 17, wherein the locations addressed are locations which aredetermined to contain at least one capture object.
 19. The method ofclaim 1, wherein the at least one signal processor is configured suchthat when the measure of the concentration of analyte molecules orparticles in the fluid sample is based at least in part on the measureindicative of the number of locations containing at least one analytemolecule or particle, the measure of the concentration is based at leastin part on a Poisson distribution analysis involving the number of thelocations associated with at least one analyte molecule or particle ofthe fluid sample.
 20. The method of claim 1, wherein the locations arereaction vessels each having a volume between about 10 attoliters and100 picoliters.